Target reporter constructs and uses thereof

ABSTRACT

Provided herein are methods and compositions for the detection of target nucleic acids using target reporter constructs (TRCs) which comprise target sequences complementary to the target nucleic acid. Further provided are methods of replicating the TRCs using rolling circle replication and/or rolling circle amplification to produce replicated TRCs which can be detected using probe sequences within the replicated TRCs.

The present application is a continuation of U.S. patent applicationSer. No. 15/614,958, filed Jun. 6, 2017, which claims the prioritybenefit of U.S. provisional application No. 62/346,334, filed Jun. 6,2016, the entire contents of which are incorporated herein by reference.

The sequence listing that is contained in the file named“REDVP0003USC1_ST25.txt”, which is 4 KB (as measured in MicrosoftWindows) and was created on Mar. 22, 2022, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns compositions and methods for thedetection of nucleic acids. More specifically, certain embodimentsconcern target reporter constructs (TRCs) for the detection of targetnucleic acids using rolling circle replication or rolling circleamplification.

2. Description of Related Art

Detection of a particular nucleic acid sequence or group of sequences isan important aspect of many biological research and clinicalapplications, particularly for diagnoses and management of medicalconditions. Methods of detecting nucleic acids, like pathogenic DNA,disease-related messenger RNAs (mRNAs) and oncogenic microRNAs (miRNAs),include reverse transcriptase polymerase chain reaction (RT-PCR), insitu microarray, and next-generation sequencing (NGS). These and otherderivative methods have been developed to detect, and in someapplications, quantify the presence of nucleic acid sequences ofinterest. However, such technologies have significant limitations andthe commercially available detection platforms that leverage suchmethods are not ideal for diagnostic analysis.

Nucleic acid detection offers significant advantage over protein-baseddiagnostic methods. For example, an immunoassay is a cheap, fast, andsensitive method for screening seroconverted individuals, but thistechnology cannot identify those individuals with recent, acuteinfection—an important population in terms of disease management. Morerecent antigen-antibody (Ag-Ab) tests target antibodies and pathogeniccomponents simultaneously but require blood or plasma as a startingmaterial, and may suffer from sensitivity and specificity issues.Furthermore, the presence of antibodies yields little informationregarding the real-time activity of the pathogen. The minimalcorrelation between viral load and antibody titer, especially in latentinfection, means that the qualitative positive-negative readout ofcurrent oral point-of-care (POC) tests gives little direction forstratification and disease management. Finally, the current oral POCdiagnostics “rule out” single pathogen systems necessitate theinconvenient use of an additional test for each target. New and diverseetiological agents in the form of bacterial, fungal, and viralinfections present significant challenges for detection, identificationand management at the POC level. Development of such technologies,particularly those using oral biospecimens, has traditionally focused onsingle-pathogen analysis with a qualitative answer; this “rule-out”approach has significant limitations and minimal utility for progressivedisease stratification.

Cell-free nucleic acids like circulating DNA, mRNA, and non-coding RNAs(ncRNAs) are powerful indicators of human health and can be used todiagnose, stratify and treat a wide range of diseases. Importantly, manynatural and pathogenic processes release nucleic acids into biofluidslike blood, urine, mucus, semen, vaginal fluid, and cerebrospinal fluidwhere minimally-invasive samples can be obtained for testing,monitoring, and treatment.

MicroRNAs (miRNAs) are short, non-coding, single-stranded RNA moleculesthat mediate cell signaling pathways by regulating the translation oftarget mRNA transcripts. Individual miRNAs have been shown to governmany important physiological and pathogenic processes, includingproliferation, cell-cycle control, apoptosis, and differentiation andare therefore important regulatory elements in transformation, tumordevelopment, and disease progression. Importantly, a large body ofevidence suggests that miRNA populations are highly dynamic,specifically with regard to neoplastic initiation and growth; the sizeand makeup of resident and circulating miRNA populations varies greatlydepending on the malignant tissue, disease stage and subtype, andbiochemical and genetic abnormalities.

Cell-free, circulating miRNAs have emerged as powerful, yetunderutilized, biomarkers in translational medicine. Plasma samplecollection is a minimally-invasive technique, and the stable,tumor-specific and clinically meaningful presence of cell-free miRNAs inplasma present certain advantages over traditional protein-basedbiomarkers that suffer from stability, sensitivity, and specificityissues. miRNAs may be present freely in the body fluids (blood, urine,saliva, mucus, etc.), bound to proteins, or present in exosomes.

Comprehensive cataloguing studies continue to define unique resident andcell-free miRNA signatures that have the ability to distinguish betweennormal and disease states, and to further differentiate individualsbased on risk, outcome and therapeutic resistance. Particularlyimportant are those cell-free miRNA signatures that representearly-detection biomarkers and might guide intervention strategies.Individual miRNA species and multi-miRNA signatures have been identifiedacross a broad spectrum of conditions, including: cancer, mentalillness, neurodegenerative and metabolic disorders, etc.

Development of cell-free miRNA profiling as a routine diagnostic tool ishindered by the lack of rapid and accessible strategies for easilycollecting, detecting, quantifying and interpreting the exceedinglyminute cell-free miRNA populations. Challenging technical issues,inter-patient variability, and the presence of multiple miRNA isoformshave further complicated widespread clinical adoption. While RT-PCR, insitu microarray, and NGS strategies have been successfully employed fordiscovery and initial characterization of cell-free miRNA populations,these approaches have limited utility in widespread screening andsurveillance applications. For example, all current methods requirerelatively large amounts of input material. The low-throughput andqualitative nature of RT-PCR and microarray results can also beproblematic when analyzing rare miRNA species. Library preparation,deep-sequencing, and bioinformatics analysis are technically demandingand expensive, making NGS-based methods impractical as a diagnosticplatform. Therefore, few commercially available diagnostic platformsexist to address the emerging need for reliable, reproducible,cost-effective and convenient quantification of circulating miRNAs.Thus, a streamlined process to functionally profile nucleic acids likepathogenic DNA, disease-related mRNA, and cell-free miRNA populationswould be a valuable addition to current disease diagnosis and treatmentstrategies.

SUMMARY OF THE INVENTION

In one aspect, there are provided target reporter constructs (TRCs) fordetecting a target nucleic acid, the TRCs being closed, partiallysingle-stranded nucleic acid molecules comprising: (a) a target sequencecomplementary to the target nucleic acid, (b) a bridge sequence forminga double-stranded portion of the TRC; and (c) an accessory sequencecomprising a multifunctional probe sequence. In some embodiments, theTRCs comprise DNA, RNA, or a combination thereof. In some embodiments,the TRCs comprise standard canonical bases, modified bases, non-naturalbases, or any combination of the bases thereof.

In some embodiments, the target nucleic acid is single-stranded DNA(ssDNA), double-stranded DNA (dsDNA), or ssRNA. In some embodiments, thessRNA is microRNA (miRNA) (e.g., cell-free miRNA). In some embodiments,the ssRNA is a non-coding RNA (ncRNA). In some embodiments, the ncRNA issmall interfering RNA (siRNA), piwi-interacting RNA (piRNA), miRNA, longnon-coding RNA (lncRNA), or small nucleolar RNA (snoRNA).

In some embodiments, the target sequence is further defined as a toeholdswitch, wherein the toehold switch is a sequence capable of uniquelyhybridizing to the target nucleic acid. In some embodiments, the toeholdswitch comprises a double-stranded switch stem and a single-strandedtoehold. In some embodiments, the toehold is between 5 and 10nucleotides in length. In some embodiments, the toehold is 7, 8, or 9nucleotides in length. In some embodiments, the toehold switch comprisesa portion of the bridge sequence. In some embodiments, the toeholdswitch is within 5-10 nucleotides of the bridge sequence. In someembodiments, the toehold switch is within 10-20 nucleotides of thebridge sequence. In some embodiments, the toehold switch is within 1-5nucleotides of the bridge sequence. In some embodiments, the toeholdswitch and bridge sequence overlap. In some embodiments, the targetsequence or toehold switch is structurally independent from the bridgesequence.

In some embodiments, the TRCs further comprise a second target sequencecomplementary to a second target nucleic acid.

In some embodiments, the bridge sequence comprises a palindromicsequence. In some embodiments, the bridge sequence is between the targetsequence and accessory sequence. In some embodiments, the bridgesequence comprises a low complexity, repetitive sequence, such as di-,tri-, and/or tetra-nucleotide repeats.

In some embodiments, the TRCs comprise multiple, such as two or more,bridge sequences. In some embodiments, the multiple bridge sequences areidentical in sequence. In some embodiments, the multiple bridgesequences comprise different sequences.

In some embodiments, the accessory sequence further comprises at least asecond probe sequence. In some embodiments, the second probe sequence isdistinct from the first probe sequence. In some embodiments, the firstprobe sequence and distinct second probe sequence form a uniquemultiplex probe signature. In some embodiments, the first probe sequenceis specific to a first detectable moiety, such as a fluorophore, and thesecond probe sequence is specific to a second detectable moiety, such asa second fluorophore. In some embodiments, the unique probe signaturefurther comprised a third, fourth, or fifth probe sequence. In someembodiments, the unique multiplex probe signature comprises a pluralityof fluorophores, such as 2, 3, 4, 5, or more fluorophores.

In some embodiments, the TRCs form a loop structure under hybridizationconditions that are well known in the art. In some embodiments, the TRCsform a hairpin structure or dumbbell structure. In some embodiments, theTRCs form a three- or four- looped structure. In some embodiments, theTRCs form a multi-loop structure. In some embodiments, the accessorysequence or portion thereof (e.g., probe sequence) forms the loopsstructure and the bridge sequence and/or the toehold switch stem formsthe stem region of a dumbbell structure.

In some embodiments, the multifunctional probe sequence comprises abinding sequence. In some embodiments, the binding sequence facilitateslocalization of replicated TRCs to an oligonucleotide array. In someembodiments, the oligonucleotide array is used to identify and quantifythe presence of target nucleic acids.

In some embodiments, the TRCs are between 50 and 100 nucleotides inlength. In some embodiments, the TRCs are between 100 and 500nucleotides in length.

In another aspect, there are provided a replicated TRC comprisingconcatenated monomer repeats of a TRC sequence. In some embodiments, thereplicated TRC is produced from rolling circle replication and/orrolling circle amplification of a TRC, such as a TRC of the aboveembodiments.

In a further aspect, there is provided a replicated nanospherecomprising a replicated TRC which has undergone intramolecularhybridization between the bridge sequences within the replicated TRC.

In another aspect, intramolecular hybridization between the bridgesequences within replicated TRCs is minimal and there is no higher-orderstructure formed upon replication or amplification of the TRC. In someembodiments, the replicated TRC may comprise a substantiallysingle-stranded product that can be detected (e.g., by probes orlocalization) or serve as a substrate for RCA reactions.

A further aspect provides a method of detecting a target nucleic acidcomprising: (a) obtaining target reporter constructs (TRCs) of theembodiments, (b) contacting the TRCs with a population of target nucleicacids, wherein individual target nucleic acid molecules within thepopulation hybridize to the complementary target sequence of thetarget-specific TRC, (c) performing rolling circle replication (RCR),thereby obtaining replicated TRCs, (d) introducing at least onedetectable moiety, wherein the at least one detectable moiety binds theprobe sequence of the replicated TRC; and (e) detecting the at least onedetectable moiety, thereby detecting the target nucleic acid.

A further aspect provides a method of detecting target nucleic acidcomprising: (a) obtaining target reporter constructs (TRCs) of theembodiments, (b) contacting the TRCs with a population of target nucleicacids, wherein individual target nucleic acid molecules within thepopulation hybridize to the complementary target sequence of thetarget-specific TRC, (c) performing rolling circle replication (RCR),thereby obtaining replicated TRCs, (d) introducing an intercalating dyethat binds the replicated TRC, and (e) detecting the detectable signalfrom such a bound dye, thereby detecting the target nucleic acid.

A further aspect provides a method of detecting target nucleic acidcomprising: (a) obtaining target reporter constructs (TRCs) of theembodiments, (b) contacting the TRCs with a population of target nucleicacids, wherein individual target nucleic acid molecules within thepopulation hybridize to the complementary target sequence of thetarget-specific TRC, (c) performing rolling circle replication (RCR) inthe presence of pH sensitive dyes, thereby obtaining replicated TRCs,and (d) detecting the fluorescent or colorimetric signal from such adye, thereby detecting the target nucleic acid.

A further aspect provides a method of detecting target nucleic acidcomprising: (a) obtaining target reporter constructs (TRCs) of theembodiments, (b) contacting the TRCs with a population of target nucleicacids, wherein individual target nucleic acid molecules within thepopulation hybridize to the complementary target sequence of thetarget-specific TRC, (c) performing rolling circle replication (RCR)obtaining replicated TRCs; and (d) introducing the replicated TRCs ontoan organized array, and (e) detecting the replicated TRCs withintercalating dyes, fluorescent probes, or by the presence of modifiednucleotides that have been incorporated into the replicated TRC, therebydetecting the target nucleic acid.

A further aspect provides a method of detecting target nucleic acidcomprising: (a) obtaining target reporter constructs (TRCs) of theembodiments, (b) contacting the TRCs with a population of target nucleicacids, wherein individual target nucleic acid molecules within thepopulation hybridize to the complementary target sequence of thetarget-specific TRC, (c) performing rolling circle replication (RCR)obtaining replicated TRCs; and (d) introducing the replicated TRCs ontoan array which has location specific binding probes capable ofhybridizing with the accessory sequence of a target-specific replicatedTRC, and (e) detecting the replicated TRCs with intercalating dyes,fluorescent probes, or by the presence of modified nucleotides that havebeen incorporated into the replicated TRC, thereby detecting the targetnucleic acid.

A further aspect provides a method of detecting target nucleic acidcomprising: (a) obtaining target reporter constructs (TRCs) of theembodiments (b) contacting the TRCs with a population of target nucleicacids, wherein individual target nucleic acid molecules within thepopulation hybridize to the complementary target sequence of thetarget-specific TRC, (c) performing rolling circle replication (RCR),(d) performing rolling circle amplification (RCA) of the replicated TRCwith bound or free secondary primers that hybridize with the accessorysequence on the replicated TRC, and (d) detecting the replicated TRCswith intercalating dyes, fluorescent probes, or by the presence ofmodified nucleotides that have been incorporated into the replicatedTRC, thereby detecting the target nucleic acid.

A further aspect provides a method of detecting target nucleic acidcomprising: (a) obtaining target reporter constructs (TRCs) of theembodiments and attaching them to a solid or porous support, such as butnot limited to a styrene bead or microscope slide (b) contacting theTRCs with a population of target nucleic acids, wherein individualtarget nucleic acid molecules within the population hybridize to thecomplementary target sequence of the target-specific TRC, (c) performingrolling circle replication (RCR), (d) performing rolling circleamplification (RCA) of the replicated TRC with bound or free secondaryprimers that hybridize with the accessory sequence on the replicatedTRC; and (d) detecting the replicated TRCs with intercalating dyes,fluorescent probes, or by the presence of modified nucleotides that havebeen incorporated into the replicated TRC, thereby detecting the targetnucleic acid.

In some embodiments of the above aspects, performing RCR and/or RCAcomprises introducing a strand-displacing polymerase, such as a DNApolymerase or RNA polymerase. In some embodiments, the DNA polymerase isφ29, Bst, Bsu, or Klenow exonuclease.

In some embodiments, performing RCR and/or RCA comprises introducing apolymerase and accessory proteins like topoisomerases, helicases,single- and double-stranded binding proteins to facilitate stranddisplacement.

In some embodiments, step (b) and step (c) of the above aspects areperformed in a single tube. In some embodiments, RCR is performed for 45minutes to 90 minutes. In some embodiments, RCR is performed for 90minutes to 48 hours.

In some embodiments, the target nucleic acids have a length greater thanthe length of the TRC, thereby producing a 3′ overhang or a 5′ overhang.In some embodiments, the method further comprises introducing anexonuclease. In some embodiments, the exonuclease is exonuclease T. Insome embodiments, the exonuclease removes a 3′ overhang of the targetnucleic acid hybridized to the TRC, thereby presenting a 3′-OH group.

In some embodiments, obtaining replicated TRCs comprises hybridizing thebridge of at least one of the replicated TRCs within the population tothe bridge of at least a second replicated TRC within the population. Insome embodiments, the replicated TRCs comprise at least two replicatedTRCs interconnected at the bridge. In some embodiments, a replicatedTRCs comprises at least 100, 1000, 5000, or 50000 replicated TRCsinterconnected at the bridge.

In some embodiments, the population of target nucleic acids comprisespathogenic nucleic acids, such as viral pathogenic nucleic acids. Insome embodiments, the pathogenic nucleic acids are humanimmunodeficiency virus (HIV), herpes simplex virus (HSV-1), humanpapillomavirus (HPV), and/or Epstein-Barr virus (EBV) pathogenic nucleicacids. In some embodiments, the target sequence for the pathogenicnucleic acids comprises a length of 50-100 nucleotides.

In some embodiments, the population of target nucleic acids comprise oneor more miRNAs. In some embodiments, the one or more miRNAs aretumor-associated miRNAs. In some embodiments, the tumor-associatedmiRNAs are selected from the group consisting of miR-let-7, miR-10b,miR-21, miR-25, miR-106b, miR-11, miR-196a , miR-210, miR-212, andmiR-221.

In some embodiments, the population of target nucleic acids comprisediabetes-associated miRNAs. In some embodiments, the diabetes-associatedmiRNAs are selected from the group consisting of miR-146a, miR-195,miR-320, miR-29, miR-192, miR-377, miR-126, miR-203, and miR-503.

In some embodiments, the population of target nucleic acids comprisepsychiatric disorders-associated miRNAs. In some embodiments, thepsychiatric disorders-associated miRNAs can be selected from the groupconsisting of miR-128, miR-134, miR-182, miR-652, miR-132, miR-15b,miR-let-7b, miR-let-7c, miR-1202, miR-135, miR-124, miR-let-7d,miR-181a, miR-212, miR-207, miR-298, miR-26b, miR-30b, miR-29b, miR-195,miR-92, miR-30a-5p, miR-30d, miR-20b, miR-29c, miR-29a, miR-106b, miR-7,miR-24, miR-30c, miR-9-3p, let-7g, miR-181b, miR-185, miR-674, miR-532,miR-673, miR-224, miR-491, miR-93, miR-383, miR-422b, miR-708, miR-540,miR-106b, miR-140, miR-194, miR-325, miR-494, miR-362, miR-409, miR-323,miR-669a, miR-151, miR-18, miR-219, miR-596, miR-597, miR-124-1,miR-598, miR-320, miR-486, miR-219, miR-346, miR-219, miR-1202, miR-135,and miR-16.

In some embodiments, the population of target nucleic acids comprisegynecological-associated miRNAs. In some embodiments, thegynecological-associated miRNAs can be selected from the groupconsisting of miR-451a, miR-20a, miR-29c, miR-145, miR-200a, miR-191,miR-543, miR-141, miR-let-7b, miR-126, miR-17, miR-210, miR-202,miR-122, miR-183, miR-196a, miR-2, miR-22, miR-let-7d, miR-143, miR-195,miR-200b, miR-21, miR-940, miR-4634, miR-100, miR-10b, miR-128, miR-1,miR-215, miR-23a, miR-143, miR-195, miR-200b, miR-21, miR-940, miR-4634,miR-100, miR-10b, miR-1215, miR-23a, miR-23b, miR-26a-1, miR-135b,miR-196b, miR-503, miR-504, miR-629, miR-10a, miR-100, miR-184,miR-193a, miR-297, miR-625, miR-let-7a/f/d/g, miR-146b , miR-5p,miR-155, miR-193a, miR-297, miR-602, miR-888, miR-212, miR-662, miR-299,miR-339, miR-5p, miR-486, miR-5p, miR-768, miR-5p, miR-376a, miR-15a,miR-29a, miR-30d, miR-93*, miR-125miR-a, miR-320a, miR-7*, miR-425,miR-744, miR-146, miR-7d, miR-202, miR-7e, miR-233b, miR-523amiR-3p,miR-188, miR-10a, miR-105, miR-182, miR-372, miR-27b, miR-339, miR-3p,miR-345, miR-25, miR-302c, miR-196a2, miR-181a, miR-372, miR-645, miR-9,miR-18b, miR-19b, miR-27b, miR-30c, miR-93, miR-103, miR-132, miR-135a,miR-146a, miR-155, miR-222, miR-224, miR-320, and miR-383.

In some embodiments, the population of target nucleic acids compriseneurodegenerative disease-associated miRNAs. In some embodiments, theneurodegenerative disease-associated miRNAs can be selected from thegroup consisting of miR-195, miR-30d, miR-451, miR-328, miR-92a,miR-486, miR-505, miR-362, miR-151, miR-20a, miR-let-7, miR-106a*,miR-133b, miR-153, miR-184*, miR-205, miR-21*, miR-224, miR-26b,miR-301b, miR-34b/c, miR-373, miR-433, miR-64, miR-65, miR-7, miR-9,miR-106a, miR-106b, miR-107 miR-124a, miR-132, miR-146a, miR-153,miR-181c, miR-29a, miR-29b, miR-29c, miR-34a, miR-429, miR-106b,miR-124a, miR-125b, miR-146a, miR-150, miR-200a, miR-200c, miR-34b,miR-9, miR-9*, miR-206, miR-21, miR-31, miR-30c-1, miR-340, miR-208b,miR-499a, miR-4538, miR-4539, miR-208a, miR-95, miR-486, miR-1, miR-539,miR-606, miR-454, miR-124a, miR-146a*, miR-206, miR-338, miR-3p, miR-9,miR-146a, miR-106b, miR-134, miR-155, miR-935, miR-149, miR-196amiR-2,miR-203a, miR-219a, miR-1, miR-301a, miR-30a, miR-34a, miR-499a,miR-876, miR-1295a, miR-10b, miR-15a, miR-193a, miR-204, miR-21,miR-27a, miR-378a, and miR-487a.

In some embodiments, the population of target nucleic acids compriseautoimmune disease-associated miRNAs. In some embodiments, theautoimmune disease-associated miRNAs can be selected from the groupconsisting of miR-146a, miR-155, miR-16, miR-101, miR-21, miR-130b, miR-146a, miR-26-1, miR-125a, miR-150, miR-638, miR-198, miR-29c, miR-30b,miR-155, miR-371a, miR-422a, miR-423, miR-410, miR-663a, miR-127,miR-221, miR-222, miR-380, miR-132, miR-141, miR-17, miR-200a, miR-30a,miR-572, miR-181c, miR-196a-2, miR-22, miR-27a, miR-499a, miR-1915,miR-106a, miR-126, miR-214, miR-320a, miR-328, miR-381, miR-422a,miR-633, miR-922, miR-let-7e, miR-let-7g, miR-106b, miR-197, miR-199a-2,miR-19a, miR-19b-1, miR-210, miR-215, miR-219a-1, miR-23b, miR-29-1,miR-9-2, miR-93, miR-338, miR-372, miR-375, miR-491, miR-146b, miR-614,miR-645, miR-648, miR-99a, miR-375, miR-424, miR-20b, miR-411, miR-629,miR-146a, miR-31, miR-29b-1, miR-122, miR-155, miR-19b-1, miR-106b,miR-203a, miR-223, miR-1246, miR-106a, miR-14, miR-200b , miR-21,miR-215, miR-29a, miR-320a, miR-595, miR-1286, miR-let-7d, miR- 107,miR- 124-1, miR-125a, miR-125b-1, miR- 126, miR- 130a, miR-148a, miR-17,miR-18a, miR-196a-2, miR-19a, miR-20a, miR-200c, miR-206, miR-23a,miR-26b, miR-30c-1, miR-9-3, miR-98, miR-155, miR-586, miR-146a,miR-214, miR-29a, miR-326, miR-199a-2, miR-26b-9, miR-3, miR-374a,miR-377, miR-423, miR-100, miR-200b, miR-30a, miR-489, miR-146b, andmiR-411.

In some embodiments, the population of target nucleic acids comprisecardiovascular disease-associated miRNAs. In some embodiments, thecardiovascular disease-associated miRNAs can be selected from the groupconsisting of miR-126, miR-210, miR-21, miR-214, miR-30d, miR-150,miR-221, miR-208a, miR-423, miR-499a, miR-208b, miR-145, miR-155,miR-22, miR-25, miR-29a, miR-29b-1, miR-340, miR-378a, miR-146a,miR-181c, miR-19b-1, miR-30a, miR-320a, miR-34a, miR-650, miR-665,miR-134, miR-137, miR-182, miR-192, miR-195, miR-199a-2, miR-199b,miR-19a, miR-223, miR-328, miR-377, miR-92b, miR-744, miR-940, miR-1292,miR-1296, miR-1825, miR-1228, miR-1293, miR-663b, miR-3148, miR-3155a,miR-3175, miR-3713, miR-4491, miR-100, miR-107, miR-10b, miR-130b,miR-142, miR-185, miR-206, miR-216a, miR-23a, miR-27b, miR-30b, miR-34b,miR-34c, miR-302c, miR-425, miR-451a, miR-146b, miR-494, miR-518e,miR-568, miR-583, miR-595 and miR-652.

In some embodiments, a single nucleic acid is the target of a TRC in asingle reaction. In some embodiments, multiple nucleic acids aretargeted in a single reaction with multiple, unique, target-specificTRCs in which each target nucleic acid is the target of atarget-specific TRC. In some embodiments, the TRCs target between 1 and2500 target nucleic acids. In some embodiments, the TRCs target between1 and 100 target nucleic acids. In some embodiments, the TRCs targetbetween 10 and 50 target nucleic acids.

In some embodiments, the TRCs are at concentrations ranging from 1zeptomolar to 1 molar in step (b). In some embodiments, the TRCs are atindividual concentrations that are dependent on the target nucleic acid.

In some embodiments, the TRCs comprise TRCs with identical targetsequences. In some embodiments, the TRCs comprise subsets of TRCs withdifferent target sequences. In some embodiments, the TRCs comprise TRCswith multiple target sequences for a viral pathogen. In someembodiments, the TRCs comprise TRCs with target sequences for more thanone viral pathogen. In some embodiments, the TRCs comprise TRCs withtarget sequences for HIV, HSV-1, and/or EBV. In some embodiments, theTRCs comprise TRCs with target sequence for more than one miRNA (e.g.,multiple miRNAs that form a miRNA signature).

In some embodiments, the method further comprises applying thereplicated TRCs of step (c) to an array prior to step (d). In someembodiments, the array is a grid-patterned array. In some embodiments,the grid-patterned array is functionalized with chemical or biologicalmoieties to direct replicated TRC positioning. In some embodiments, thechemical moieties rely on hydrophobic, hydrophilic, and/or electrostaticforces to position replicated TRCs on the grid patterned array. In someembodiments, the grid-patterned array is silanized by an aminosilane. Insome embodiments, the grid patterned array is functionalized with epoxysilane. In some embodiments, the grid patterned array is functionalizedwith isothiocyanate. In some embodiments, the grid patterned array isfunctionalized with aminopropyl-derivatized or aminophenyl. In someembodiments, the grid patterned array is functionalized withmercaptosilane. In some embodiments, the grid patterned array isfunctionalized with aldehyde or epoxide.

In some embodiments, the detectable moiety is a fluorophore,chromophore, or radioisotope. In some embodiments, the detectable moietyis soluble or insoluble. In some embodiments, the detectable moiety is afluorophore. In some embodiments, detecting the at least one detectablemoiety comprises normalizing to a control replicated TRC. In someembodiments, step (d) comprises introducing 2, 3, or 4 distinctdetectable moieties. In some embodiments, step (e) comprises detectingthe 2, 3, or 4 distinct detectable moieties. In some embodiments,detecting the 2, 3, or 4 distinct detectable moieties is further definedas detecting the unique multiplex probe signature of a replicated TRC.In some embodiments, each unique multiplex probe signature comprises aplurality of fluorophores (e.g., 2, 3, 4, 5, or more). In someembodiments, detecting a multiplex probe signature comprises hybridizinga first detectable moiety, detecting the first detectable moiety, andwashing of the array followed by hybridizing a second detectable moiety,and detecting the second detectable moiety, such that the combination ofthe first and second detectable moieties identifies a unique multiplexprobe signature. In some embodiments, detecting is performed using anepifluorescence microscope.

In some embodiments, detecting is performed using fluorometric orcolorimetric analysis of the RCR or RCA reaction, such as but notlimited to pH sensitive dyes.

In some embodiments, detecting is performed by detecting the presence ofmodified nucleotides which have been incorporated into the replicatedTRC. In some embodiments, these modified nucleotides includefluorescently labeled nucleotides, such as but not limited to Cy3, Cy5,fluorescein, or other fluorescent dyes described herein.

In some embodiments, replicated TRCs are applied to arrays and detectedby modified nucleotides or by dye staining (e.g., Sybr).

In some embodiments, replicated TRCs are applied to arrays and bind tospecific locations based on the accessory sequence which can then beused to detect and quantify the target nucleic acid(s).

In some embodiments, replicated TRCs are detected using the methodsdescribed herein using a plate reader, RT-PCR instrument, or flowcytometer.

In some embodiments, TRCs are bound to solid supports like slides orbeads, and RCR and/or RCA produces replicated TRCs that can be detectedusing the methods described herein.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The invention may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-1F: (A) Schematic of exemplary configurations for TRCconstruction with relative placement of functional sequences. (B)Schematic of TRC architecture and platform for use of TRCs to detecttarget nucleic acids. (C) Schematic of toehold switch mechanism. (D)Schematic of φ29 3′ exonuclease activity removing 3′ overhangingnucleotides up to the double-stranded complimentary region. Rollingcircle replication is initiated, creating probable replicated TRCs. (E)Schematic of titration series of synthetic oligonucleotides used toanalyze TRC metrics with fluorescent probes. Each viral pathogen hasdistinct fluorophore color. (F) Schematic of pathogenic nucleic acidisolation from diagnostic viral mimics in donor plasma. The sequencespecific TRCs hybridize to the target nucleic acid, thereby initiatingRCR and producing replicated TRCs that may be probed withpathogen-specific fluorescent probes. The digital identification ofreplicated TRCs occurs on a functionalized array. Each replicated TRChas a distinct fluorophore color.

FIGS. 2A-2C: (A-B) Sensitivity and specificity of miR-let-7a andmiR-let-7g TRCs. (C) Detection of miR-let-7a using a TRC on a qRT-PCRinstrument and Sybr reagents.

FIG. 3: Microvascular complications in Type I Diabetes: Complicationsare presented with Type I diabetes specific data for the United Statesincluding the number of patients at a given time, overall lifetime riskof development and annual healthcare expenditures. Target miRNAs arelisted for each complication along with three altered microvascularmiRNA targets.

FIGS. 4A-4B: (A) Sequence and structure of miR-let-7a TRC (SEQ ID NO: 1)is depicted. (CTACTACCT (SEQ ID NO: 2): Toehold; CGAACTATACAAC (SEQ IDNO: 3): Switch Stem, one side; C: (de)stabilizing elements/other fillerswith internal functionality; CCTACCTCCACATCCTCCACAAGCTATCCC (SEQ ID NO:4): Probe(s) or other externally functional sequences). (B) Sequences ofTRCs for miR-let-7g (SEQ ID NO: 5) and TRC for miR-21 (SEQ ID NO: 6).

FIG. 5: Sequence and structure of a multi-looped miR-21 TRC (SEQ ID NO:7).

FIG. 6: TRCs bound to solid support triggering RCR and RCA. Detected byintercalating dye.

FIGS. 7A-C: (A) Endometriosis associated miRNAs. (B) TRC validationmethods using microplate- and qRT-PCR-based methods. qRT-PCR basedapproach for quantitation of miRNAs using TRCs. (C) Endometriosisinduced mouse model, validation approach for an endometriosis miRNAsignature, and idealized data showing a correlation between plasma andtissue derived miRNA.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The dynamic and disease-specific nature of cell-free microRNA (miRNA)populations makes them a novel class of biomarkers that have shownpromise during initial studies with potentially significant clinicalutility. There are, however, very few effective and accessibletechnologies that fully leverage the growing library of informativemiRNA signatures or meet the stringent requirements necessary forwidespread adoption as diagnostic platforms. Importantly, the few teststhat have been approved by the FDA for this purpose rely on costly andtime-consuming technologies that have limited throughput, minimalflexibility, and negligible risk assessment.

Accordingly, in some aspects, the present disclosure provides rapid,robust, cost-effective, and broadly enabling platforms for the detectionof target nucleic acids. Embodiments of the present disclosure provideTRCs and methods of using these TRCs for the detection and quantitationof target nucleic acids. The nucleic acids can be pathogenic nucleicacids, such as of bacterial, fungal, and viral nucleic acids, or miRNAs,including tumor-associated miRNAs and diabetes-associated miRNAs. Othernucleic acids that can be identified include, but are not limited to,siRNA, snoRNA, lncRNA, piRNA, or full-length or fragmentedoligonucleotides with known ends either naturally or artificiallycreated (e.g., for example, enzymatically digested, CRISPR/Cas9, or useof modified nucleobases).

The TRC platform eliminates library preparation and consolidatesanalysis into a single, multiplex reaction resulting a reduced overallworkflow and lowers technical requirements. This platform necessitatesvery little hands-on time or expert analysis, and the rapid andautomated digital analysis renders usable data on a timescale necessaryfor making important health decisions.

The TRCs are rigorously designed synthetic nucleic acid (e.g., DNA)structures capable of detecting target nucleic acids, such as pathogenicnucleic acids and miRNA species, with exquisite sensitivity andspecificity. TRCs can be easily synthesized and employ a number ofunique elements and approaches. A TRC comprises a target sequencecomplementary to the target nucleic acid. In some aspects, the TRCscomprise self-complementary bridge sequences which can encourage tightlyintertwined, but non-entangled DNA structures that permit detection ofdiscrete replicated products, referred to herein as replicated TRCs. TheTRC can also comprise an accessory sequence, such as a probe sequence.In some embodiments, multiple probe-hybridization sequences facilitatethe decoding of replicated TRCs. In some embodiments, an accessorysequence can be used to localize replicated products by hybridizationfor identification and quantitation applications. Optical detection ofreplicated TRCs permits unparalleled multiplexibility to target multiplemiRNA species in a single reaction tube. The ability to incorporateadditional probe sites and more complex decoding strategies providesenormous scalability and adaptability, thus, new miRNA targets areeasily incorporated. The ability to use diverse detection strategiesincluding the incorporation of modified nucleotides in replicated TRCsand the use of intercalating dyes or colorimetric reagents makes theassay flexible for diverse applications and the ability to run the assayon many instruments (e.g., RT-PCR, plate reader, bead-based approaches,and a scanning microscope) making it accessible to a range of equippedlaboratories.

Additionally, the TRC can comprise a toehold switch which forms a shortduplex structure with an overhang for the target miRNA to hybridize toand initiate toehold-mediated strand displacement (TMSD), anenzyme-free, entropically-driven nucleic acid hybridization process thatis more specific than Watson-Crick base-pairing and padlock-probe basedapproaches. The unique nature of TMSD allows for discrimination ofsingle-nucleotide variation within miRNAs as well as detection ofdifferent miRNA isoforms.

The miRNAs function as in situ derived primers for rolling circlereplication or amplification, which effectively eliminates samplepreparation, inefficient library manipulation steps, and minimizesreaction biases that are present in nearly all other detection methods.This significantly reduces sample input volumes (from 400 μL to <10 μL),assay costs (from thousands to hundreds of dollars), and assay time(<two [2] hours). These factors enable replicate experiments to be runfor each sample, a requirement for reliable quantitation not afforded bymany previous methods. Plasma derived miRNA sequences are interrogatedin a multiplex, single tube format; this eliminates the need todistribute precious samples into multiple reaction chambers. Thisapproach further reduces the input sample size, minimizes waste, reducesbias and limits reproducibility errors that arise when rare cell-freemiRNAs are divided and distributed, as in other digital quantificationmethods. The TRC reaction may be multiplexed, targeting multiple nucleicacids in a single reaction chamber or specific to a single target perreaction.

The toehold switches also dramatically increase the specificity oftarget detection with competitive strand displacement. Other commercialsystems rely on simple complimentary hybridization, which is inherentlyvulnerable to off-target and promiscuous binding, leading tofalse-positives and unreliable data. The independent and universalreplication of all TRC-bound miRNAs eliminates amplification biastraditionally seen in PCR-based approaches.

Accordingly, in certain embodiments, the TRCs are dumbbell-shaped andcomprise three principle domains: (i) a toehold-domain capable ofuniquely hybridizing to a single complimentary miRNA, (ii) bridgingsequences to encourage the formation of intertwined DNA nanospheresduring replication, and (iii) multiple probing sites, to facilitatemultiplex fluorescent interrogation of each replicated product.

In certain embodiments, the accessory sequences can be used for probing,localization or priming for RCA.

In addition, rolling circle replication is used for isothermalamplification of the TRCs. Unlike other digital methods such as digitalPCR that relies on distributing target sequences from hundreds to tensof thousands of individual wells, the TRC assay replicates all targetsequences in a single tube format. The platform leveragesstrand-displacing properties of DNA polymerases to achieve rapid androbust replication with little temperature modulation. Persons skilledin the art will recognize strand-displacing polymerases withnon-limiting examples such as φ29, Bsu, or Bst DNA polymerase.Single-molecule detection is achieved through the stochastic arrangementof replicated TRCs on high-density ordered arrays. This strategyprovides an organized, quantifiable readout through high resolutionfluorescent imaging of the sequentially probed replicated TRCs. TRCscustom-designed bridge sequences transform the replicated products intotightly intertwined, but non-entangled DNA structures, called replicatedTRCs. Successfully replicated TRCs can then be rapidly organized onhigh-density patterned arrays for easy identification and quantitation.

Thus, embodiments of the present disclosure provide a novel multiplexdiagnostic platform to precisely characterize pathogenic nucleic acidsand cell-free miRNA signatures, specifically those miRNAs of immediateutility in diagnosis, stratification, and intervention in cancer, type IDiabetes microvascular complications, and endometriosis. The methodsprovided herein can provide fundamental information to positively affectdisease management and patient survival.

I. DEFINITIONS

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%, preferably below 0.01%. Most preferred isa composition in which no amount of the specified component can bedetected with standard analytical methods.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

A “nucleic acid molecule” or “nucleic acid sequence” refers to anysingle-stranded or double-stranded nucleic acid molecule includingstandard canonical bases, hypermodified bases, non-natural bases, or anycombination of the bases thereof. For example and without limitation,the nucleic acid molecule contains the four canonical DNA bases—adenine,cytosine, guanine, and thymine, and/or the four canonical RNAbases—adenine, cytosine, guanine, and uracil. Uracil can be substitutedfor thymine when the nucleoside contains a 2′-deoxyribose group. Thenucleic acid molecule can be transformed from RNA into DNA and from DNAinto RNA. For example, and without limitation, mRNA can be created intocomplementary DNA (cDNA) using reverse transcriptase and DNA can becreated into RNA using RNA polymerase. “Analogous” forms of purines andpyrimidines are well known in the art, and include, but are not limitedto aziridinylcytosine, N⁴-acetylcytosine, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine, N⁶-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N⁶-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, and2,6-diaminopurine. The nucleic acid molecule can also contain one ormore hypermodified bases, for example and without limitation,5-hydroxymethyluracil, 5-hydroxyuracil, α-putrescinylthymine,5-hydroxymethylcytosine, 5-hydroxycytosine, 5-methylcytosine,5-methylcytosine, 2-aminoadenine, α-carbamoylmethyladenine,N⁶-methyladenine, inosine, xanthine, hypoxanthine, 2,6-diaminopurine,and N⁷-methylguanine. The nucleic acid molecule can also contain one ormore non-natural bases, for example and without limitation,7-deaza-7-hydroxymethyladenine, 7-deaza-7-hydroxymethylguanine,isocytosine (isoC), 5-methylisocytosine, and isoguanine (isoG). Thenucleic acid molecule containing only canonical, hypermodified,non-natural bases, or any combinations the bases thereof, can alsocontain, for example and without limitation where each linkage betweennucleotide residues can consist of a standard phosphodiester linkage,and in addition, may contain one or more modified linkages, for exampleand without limitation, substitution of the non-bridging oxygen atomwith a nitrogen atom (i.e., a phosphoramidate linkage, a sulfur atom(i.e., a phosphorothioate linkage), or an alkyl or aryl group (i.e.,alkyl or aryl phosphonates), substitution of the bridging oxygen atomwith a sulfur atom (i.e., phosphorothiolate), substitution of thephosphodiester bond with a peptide bond (i.e., peptide nucleic acid orPNA), or formation of one or more additional covalent bonds (i.e.,locked nucleic acid or LNA), which has an additional bond between the2′-oxygen and the 4′-carbon of the ribose sugar. The term“2′-deoxyribonucleic acid molecule” means the same as the term “nucleicacid molecule” with the limitation that the 2′-carbon atom of the2′-deoxyribose group contains at least one hydrogen atom. The term“ribonucleic acid molecule” means the same as the term “nucleic acidmolecule” with the limitation that the 2′-carbon atom of the ribosegroup contains at least one hydroxyl group.

The nucleic acids of the present disclosure may be single-stranded ordouble-stranded, as specified, or contain portions of bothdouble-stranded or single-stranded sequence. The nucleic acids may beDNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acidcontains any combination of deoxyribo- and ribonucleotides, and anycombination of bases, including uracil, adenine, thymine, cytosine,guanine, inosine, xathanine hypoxathanine, isocytosine, and isoguanine.Two nucleic acids or nucleic acid regions are “complementary” to oneanother if they base-pair with each other to form a double-strandednucleic acid molecule.

The term “target nucleic acid” refers to a nucleic acid molecule oninterest or nucleic acid sequence on a single-strand of nucleic acid.The target sequence may be a portion of a gene, a regulatory sequence,genomic DNA, cDNA, or RNA including mRNA and rRNA. As is outlinedherein, the target nucleic acid may be a target sequence from a sample,or a secondary target such as a product of an amplification reaction. Itmay be of any length. In particular aspects, the target nucleic acid isa pathogenic nucleic acid or miRNA.

The term “microRNA (miRNA)” refers to single-stranded RNA moleculesgenerally of about 21-23 nucleotides in length, but may be 16-25nucleotides in length, which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed but miRNAs are nottranslated into protein (i.e., non-coding RNAs). The genes encodingmiRNAs are much longer than the processed mature miRNA molecule; miRNAsare first transcribed as primary transcripts or pri-miRNA with a cap andpoly-A tail and processed to short, 70-nucleotide stem-loop structuresknown as pre-miRNA in the cell nucleus. This processing is performed inanimals by a protein complex known as the Microprocessor complex,consisting of the nuclease Drosha and the double-stranded RNA bindingprotein Pasha. These pre-miRNAs are then processed to mature miRNAs inthe cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC).When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC. Thisstrand is known as the guide strand and is selected by the argonauteprotein, the catalytically active RNase in the RISC, on the basis of thestability of the 5′ end. The remaining strand, known as the miRNA*,anti-guide or passenger strand, is degraded as a RISC substrate.Therefore the miRNA*s are derived from the same hairpin structure likethe “normal” miRNAs. So if the “normal” miRNA is then later called the“mature miRNA” or “guided strand,” the miRNA* is the passenger strand.

The term “closed” refers to a single or double-stranded nucleic acidmolecule that lacks a free end. In some embodiments, closed nucleicacids may have an undefined shape and topology. In some embodiments, theclosed nucleic acid forms a secondary structure under buffer andhybridization conditions well known in the art.

“Target report constructs (TRCs)” are referred to herein as closed,single-stranded nucleic acid molecules. In some embodiments, the TRCsare molecules used to contact and detect a target nucleic acid. In someembodiments, the TRC is circular. In some embodiments, the TRC cancomprise a “dumbbell” structure meaning a structurally linear, andtopologically circular in vitro replication competent or in vitrocompetent nucleic acid molecule that has one or more hairpin structures.When denatured or substantially denatured, dumbbell structures exist ascircular, single-stranded nucleic acid molecules. The TRCs form adumbbell or hairpin structure under conditions (e.g., pH, buffer, andtemperature) known in the art.

A “hairpin structure” refers to a nucleic acid molecule whereby two ormore partial sequences within the nucleic acid molecule arecomplementary or substantially complementary to each other resulting inthe formation of a partially double-stranded region and one or moreinternal single-stranded regions. Hairpin structures can be formed byintramolecular base-pairing of complementary nucleotides orsubstantially complementary nucleotides of a given nucleic acidmolecule, which can form a stem-loop structure. The stem portion of thehairpin structure is formed by hybridization of the complementarynucleotide or substantially complementary nucleotide sequences to form adouble-stranded helix stretch. The loop region of the hairpin structureis the result of an unpaired stretch of nucleotide sequences. Thestability of the hairpin structure is dependent on the length, bufferconditions, temperature, nucleic acid sequence composition, and degreeof base-pair complementary or substantial complementary of the stemregion. For example, a stretch of five complementary nucleotides may beconsidered more stable than a stretch of three complementary nucleotidesor a stretch of complementary nucleotides that are predominatelycomposed of guanines and cytosines may be considered more stable than astretch of complementary nucleotides that are predominately composed ofadenines and thymines (DNA) or uracils (RNA). Modified nucleotides maybe substituted to alter the stability of the double-stranded stem regionfor these natural bases, examples of which include, but are not limitedto, inosine, xanthine, hypoxanthine, 2,6-diaminopurine,N⁶-methyladenine, 5-methylcytosine, 7-deazapurines,5-hydroxylmethylpyrimidines. Modified nucleotides may also includenumerous modified bases found in RNA species. Natural occurringstem-loop structures are predominately found in RNA species, such astransfer RNA (tRNA), pre-microRNA, ribozymes and their equivalents.

A “toehold switch” refers to a hairpin structure within the TRC whichcomprises a sequence fully or partially complimentary to the targetnucleic acid. The toehold switch may be positioned so that the targetnucleic acid binds partially within the loop and partially within stemregions. As such, the binding of a target nucleic acid may open thetoehold switch, permitting RCR.

A “bridge sequence” refers to two or more sequences within a nucleicacid molecule that are self-complimentary or substantiallyself-complimentary. In some embodiments, in certain TRC architectures,the bridge sequences are complimentary and hybridize under certainconditions to generate a secondary structure within the molecule. Incertain embodiments, in some replicated TRCs, a bridge sequence mayhybridize with one or more additional bridge sequences generatedfollowing RCR or RCA.

The term “self-complimentary” as used herein refers to a nucleic acidmolecule with one or more complimentary or substantially complimentarysequences contained along its length capable of intramolecularhybridization. In some embodiments, two nearly adjacentself-complimentary sequences separated by as few as one base-pair mayhybridize. In some embodiments, the self-complimentary sequences may beseparated by 10 to 100 base-pairs. In some embodiments, such as but notlimited to certain TRC architectures, the self-complimentary sequencesmay be separated by an accessory sequence, target sequence or both.

The term “accessory sequence” as used herein refers to a uniquelydesigned component sequence of a TRC that may be used to detect thereplicated TRC following RCR and/or RCA. In some embodiments, theaccessory sequence may comprise a uniquely designed probe sequence towhich a complimentary or substantially complimentary fluorescent probewill bind the replicated TRC, permitting detection of the target nucleicacid. In some embodiments, the accessory sequence comprises a uniquelydesigned sequence which can be used to localize replicated TRCs to aspecific spot on an array, permitting detection of a target nucleicacid. In some embodiments, the accessory sequence may be used for asingle purpose. In some embodiments, the accessory sequence may be usedfor multiple detection approaches, such as a fluorescent probe,localization, or a combination thereof, and is thus termed herein as a“multifunctional probe sequence.”

Nucleic acid hairpin structures may be generated by deliberate designusing methods of manufacturing synthetic oligonucleotides.Oligonucleotides are widely used as primers for DNA sequencing and PCR,as probes for screening and detection experiments, and as linkers oradapters for cloning purposes. Short oligonucleotides in the range of 15to 25 nucleotides can be used directly without purification. As thestepwise yields are less than 100%, longer oligonucleotides requirepurification by high performance liquid chromatography or HPLC, or bypreparative gel electrophoresis to remove failed oligonucleotidefractions, also known as n-1, n-2, etc. products. In certainembodiments, the nucleic acid hairpin is approximately about 100 bases.

Depending on the nature of the experiment, a given hairpin structure maybe designed to contain a desired stability of the double-stranded duplexby substituting one or more hypermodified or non-natural bases and/orone or more backbone linkages as discussed herein, or including othersynthetic bases such as 7-deaza-7-hydroxypurines, isoC and isoG, ortheir equivalents, as well as creating, for example, and withoutlimitation, RNA-DNA, PNA-DNA, PNA-RNA, PNA-PNA, LNA-DNA, LNA-RNA,LNA-LNA, and double-stranded duplexes. Synthetically-designed hairpinstructures are useful in several molecular biology techniques, forexample, and without limitation, as priming sites for DNA polymerase byligating hairpins to the ends of DNA fragments, detecting moieties asprobes to identify a sequence of interest, and creating topologicallycircular DNA molecules from linear fragments. In certain embodiments,the 5′-ends of one or more hairpin structures will be phosphorylated,for example and without limitation, using T₄ polynucleotide kinase tofacilitate the efficient ligation using ligating agents to the ends ofone or more fragmented nucleic acid molecules.

Hairpin structures have also been used as oligonucleotide probes. DNAprobes, also known as molecular beacons, are oligonucleotides designedto contain an internal probe sequence with two ends that arecomplementary to one another. Under appropriate conditions, the endshybridize together forming a stem-loop structure. The probe sequence iscontained within the loop portion of the molecular beacon and isunrelated to the stem arms. A fluorescent dye is attached to one end onthe stem and a non-fluorescent quenching moiety or “quencher” isattached to the other end of the stem. In the stem-loop configuration,the hybridized arms keep the fluorescent dye and quencher in closeproximity, resulting in quenching of the fluorescent dye signal by thewell-understood process of fluorescence resonance energy transfer(FRET). When the probe sequence within the loop structure finds andhybridizes with its intended target sequence, the stem structure isbroken in favor of the longer and more stable probe-target duplex. Probehybridization results in the separation of the fluorescent dye andquencher (i.e., the close proximity is now lost), for which dye can nowfluoresce when exposed to the appropriate excitation source of thedetector. Molecular beacons have been used in a number of molecularbiology techniques, such as RT-PCR, to discriminate allelic differences.

In certain embodiments, the hairpin structures can be created by usingtwo or more nucleic acid molecules that are then joined to form a singlehairpin structure. The two or more nucleic acid molecules can be joinedtogether using ligating reagents to form a hairpin structure. The two ormore nucleic acid molecules can also be chemically joined together usinga linker to form a hairpin structure. In certain embodiments, the5′-ends of one or more hairpin structures will be phosphorylated, forexample and without limitation, using T₄ polynucleotide kinase tofacilitate the efficient ligation using ligating agents to the ends ofone or more fragmented nucleic acid molecules.

In certain embodiments, functionally important information can reside inthe stem region of the hairpin structure. In certain embodiments,functionally important information can reside in the loop region of thehairpin structure. Functionally important information can include, forexample and without limitation, the necessary sequences for in vitroreplication, in vitro amplification, unique identification (i.e.,barcodes), and detection. In certain embodiments where the functionallyimportant information resides in the loop region of the hairpinstructure, the length of the stem region can be as few as four or sixbase-pairs. In certain embodiments where the functionally importantinformation resides in the stem region of the hairpin structure, thelength of the loop region can be as few as one or two bases.

“Contacting” means a process whereby a substance is introduced by anymanner to promote an interaction with another substance. For example,and without limitation, a dumbbell template may be contacted with one ormore substantially complementary primers to promote one or morehybridizing processes to form one or more double-stranded duplex regionscapable of participating in rolling circle replication or rolling circleamplification.

“Detecting a nucleic acid molecule” means using an analytical methodthat can determine the presence of the nucleic acid of interest or thatcan determine more detailed information regarding the nucleic acidsequence, alterations of a nucleic acid sequence when compared with areference sequence, or the presence or absence of one or more copies ofthe nucleic acid sequence.

“End(s) of a fragmented nucleic acid molecule(s)” means one or moreterminal nucleotide residues capable or to be made capable ofparticipating in a ligation reaction. In certain embodiments, one ormore nucleic acid molecules may contain functional ends capable or to bemade capable of a ligation reaction to attach one or more hairpinstructures to each end of the nucleic acid molecule. For example, andwithout limitation, the 5′-end terminal nucleotide contains a phosphategroup and the 3′-end terminal nucleotide contains a hydroxyl group.

“Isolating a nucleic acid molecule” means a process whereby a nucleicacid molecule is obtained from a sample.

“Linker” means one or more divalent groups (linking members) thatfunction as a covalently-bonded molecular bridge between two othernucleic acid molecules. A linker may contain one or more linking membersand one or more types of linking members. Exemplary linking membersinclude: —C(O)NH—, —C(O)O—, —NH—, —S—,—S(O)n—where n is 0, 1,or 2, —O—,—OP(O)(OH)O—, —OP(O)(O⁻)O—, alkenediyl, alkenediyl, alkynediyl,arenediyl, heteroarenediyl, or combinations thereof. Some linkers havependant side chains or pendant functional groups (or both). Pendantmoieties can be hydrophilicity modifiers (i.e., chemical groups thatincrease the water solubility properties of the linker), for example andwithout limitation, solubilizing groups such as —SO₃H, —SO₃ ⁻, CO₂H orCO₂ ⁻.

“Performing” means providing all necessary components, reagents, andconditions that enable a chemical or biochemical reaction to occur toobtain the desired product.

“Purifying” means removing undesired nucleic acid molecules that did notsuccessfully ligate to form dumbbell templates for any given size range.

The term “complementary” is used herein to mean that at least 80% of thebases undergo Watson-Crick base-pairing, such as 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% of the bases undergo Watson-Crickbase-pairing. For example, a complementary sequence may comprise up to20%, such as 10% or 5%, of bases which are not Watson-Crick base-paired.

“Substantially complementary primer” means a nucleic acid molecule thatforms a stable double-stranded duplex with another nucleic acidmolecule, although one or more bases of the nucleic acid sequence withinthe duplex region do not base-pair(s) with the another nucleic acidsequence.

“Rolling circle replication (RCR)” refers to a unidirectional nucleicacid replication process that can synthesize multiple copies of circularnucleic acids, such as TRCs. In some embodiments, the target nucleicacid serves as the primer for RCR of a TRC with a complementary targetsequence to generate multiple copies of the TRC in a continuous seriescalled a “concatemer”. The result of RCR from a target primed TRC is a“replicated TRC.”

“Rolling circle amplification (RCA)” refers to the amplification processby which the product of initial RCR serves as a substrate and is primedone or more times by a second primer to generate multiple copies of theRCR product.

A “replicated TRC” refers to a multimeric nucleic acid moleculecomprising concatenated monomer repeats of complimentary TRC sequences.In particular embodiments, a replicated TRC is produced from rollingcircle replication or rolling circle amplification of a TRC. Inparticular embodiments, the replicated TRC undergoes intermolecularhybridization between bridge sequences, forming a “replicatednanosphere”.

The term “primer” or “oligonucleotide primer” as used herein, refers toan oligonucleotide that hybridizes to the template strand of a nucleicacid and initiates synthesis of a nucleic acid strand complementary tothe template strand when placed under conditions in which synthesis of aprimer extension product is induced, i.e., in the presence ofnucleotides and a polymerization-inducing agent such as a DNA or RNApolymerase and at suitable temperature, pH, metal concentration, andsalt concentration. The primer is generally single-stranded for maximumefficiency in amplification, but may alternatively be double-stranded.If double-stranded, the primer can first be treated to separate itsstrands before being used to prepare extension products. Thisdenaturation step is typically effected by heat, but may alternativelybe carried out using alkali, followed by neutralization. Thus, a“primer” is complementary to a template, and complexes by hydrogenbonding or hybridization with the template to give a primer/templatecomplex for initiation of synthesis by a polymerase, which is extendedby the addition of covalently bonded bases linked at its 3′ endcomplementary to the template in the process of DNA or RNA synthesis. Inparticular embodiments, the target nucleic acid, such as a miRNA in asample, is utilized as the primer for RCR.

The term “label” or “probe sequence” as used herein means a molecule ormoiety having a property or characteristic which is capable ofdetection. A label or probe sequence can be directly detectable, aswith, for example, radioisotopes, fluorophores, chemiluminophores,enzymes, colloidal particles, and fluorescent microparticles; or a labelmay be indirectly detectable, as with, for example, specific bindingmembers. It will be understood that directly detectable labels mayrequire additional components such as, for example, substrates,triggering reagents, and light to enable detection of the label. Whenindirectly detectable labels are used, they are typically used incombination with a “conjugate.” A conjugate is typically a specificbinding member which has been attached or coupled to a directlydetectable label. Coupling chemistries for synthesizing a conjugate arewell known in the art and can include, for example, any chemical meansand/or physical means that does not destroy the specific bindingproperty of the specific binding member or the detectable property ofthe label. As used herein, “specific binding member” means a member of abinding pair, i.e., two different molecules where one of the moleculesthrough, for example, chemical or physical means specifically binds tothe other molecule. In addition to antigen and antibody specific bindingpairs, other specific binding pairs include, but are not intended to belimited to, avidin and biotin; haptens and antibodies specific forhaptens; complementary nucleotide sequences; and enzyme cofactors orsubstrates and enzymes.

“Sample” means a material obtained or isolated from a fresh or preservedbiological sample or synthetically-created source that contains anucleic acid molecule of interest. In certain embodiments, a sample isthe biological material that contains the desired nucleic acid for whichdata or information are sought. Samples can include at least one cell,fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva,urine, tear, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid,mucosa secretion, peritoneal fluid, ascites fluid, fecal matter, bodyexudates, umbilical cord blood, chorionic villi, amniotic fluid,embryonic tissue, multicellular embryo, lysate, extract, solution, orreaction mixture suspected of containing a target nucleic acid molecule.Samples can also include non-human sources, such as non-human primates,rodents and other mammals, plants, pathogenic species including viruses,bacteria, and fungi. In certain embodiments, the sample can also includeisolations from environmental sources for the detection of human andnon-human species as well as pathogenic species in blood, water, air,soil, food, and for the identification of all organisms in the samplewithout any prior knowledge. In certain embodiments, the sample maycontain nucleic acid molecules that are degraded. Nucleic acid moleculescan have nicks, breaks or modifications resulting from exposure tophysical forces, such as shear forces, to harsh environments such asheat or ultraviolet light, to chemical degradation processes such as maybe employed in clinical or forensic analyses, to biological degradationprocesses due to microorganisms or age, to purification or isolationtechniques, or a combination thereof.

The basic structure of single-stranded and double-stranded nucleic acidmolecules is dictated by base-pair interactions. For example, theformation of base-pairs between complementary or substantiallycomplementary nucleotides on the two opposite strands will cause the twostrands to coil around each other to form a double-helix structure. Thisis called intermolecular base-pairing of complementary nucleotides oftwo or more nucleic acid molecule strands. The term “nucleotide” isdefined broadly in the present disclosure as a unit consisting of asugar, base, and one or more phosphate groups, for which the sugar, forexample, and without limitation, consists of a ribose, a modified ribosewith additional chemical groups attached to one or more atoms of theribose group, a 2′-deoxyribose, or a modified 2′-deoxyribose withadditional chemical groups attached to one or more atoms of the2′-deoxyribose group, and for which the base, for example, and withoutlimitation, consists of a canonical base, hypermodified base, ornon-natural base, as described in the nucleic acid molecule definitionabove. Base-pairing of complementary nucleotides or substantiallycomplementary nucleotides can also occur on the same DNA strandmolecule, called intramolecular base-pairing of complementarynucleotides or substantially complementary nucleotides.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

I. Target Reporter Constructs

Certain aspects of the present disclosure concern target reporterconstructs (TRCs) for single-molecule detection of target nucleic acids,such as cell-free miRNAs and pathogenic nucleic acids. The TRCs arerigorously designed, complex nucleic acid sequence structures thatcomprise a target sequence which is complimentary to the target nucleicacid, bridge sequences to encourage the formation of nano spheres duringreplication, and probing sequences to facilitate multiplex fluorescentinterrogation of each TRC-target nucleic acid replicated product asoutlined in FIG. 1. In some embodiments, the probing sequence maycomprise accessory sequences that permit detection by other methods. Insome embodiments, a TRC can comprise a length of about 10-500nucleotides, 50-100 nucleotides, 20-40 nucleotides, 75-150 nucleotides,or 10-50 nucleotides.

The design of the TRCs may comprise a bioinformatics approach employingthe sequence alignment tool, BLAST, and the small genome alignment tool,HMMer, to generate an E-value matrix that compares selected genomes in asliding window fashion. For example, candidate pathogen sequences thatmeet a heuristically determined E-value threshold are analyzed toconsider optimal sequence length, T_(m), and GC content to fully assesstheir “targetability,” and inclusion in the TRC library.

One exemplary method for constructing a TRC comprises usingsingle-stranded DNA oligonucleotides (e.g., Integrated DNATechnologies). Multiple TRCs are generated specific for each miRNA withthe final miRNA-specific TRCs (one per miRNA target) selected afterempirical testing. The 5′ and 3′ ends of the oligonucleotide arestrategically placed within the toehold or bridge sequences; slowcooling under high-salt conditions forces the 5′ and 3′ ends to sitadjacent and encourages the correct intra-molecular ligation by T4ligase (New England Biolabs). Treatment with exonucleases I and IIIeliminate unligated reactants. Gel electrophoresis is used to confirmthe correct size and purity of the final TRC population.

In some embodiments, synthetic RNA oligonucleotides designed to mimictarget and non-target miRNAs are used to establish and optimize thespecificity and sensitivity of constructed TRCs. Reaction temperature,duration, buffer conditions, and reagent concentrations may be varied inan effort to define optimal conditions at each stage of the librarycreation process. Individual TRCs can be validated using multiplecriteria. For example, a titration series of the target miRNA is usedinitially to gauge basic sensitivity and the ability to initiate RCR DNAsynthesis. Second, mimic miRNAs with varying degrees of mismatch to thetarget miRNA sequence are used to quantify specificity of the individualTRC. Finally, a titration series of the target miRNA over a non-specificmiRNA background is used to assess the robust ability of the TRC tofunction in diagnostic-like setting. All individual TRC reactions may bequantified with a molecular-beacon approach, with reactions beingmonitored by qRT-PCR and final products by microplate.

A. Target Sequence

A TRC of present disclosure comprises one or more target sequences whichare complementary to one or more target nucleic acids. Design of thetarget sequences can be achieved through extensive cross-alignmentbetween targeted and non-targeted sequences in order to minimize falsepositives and obtain robust, specific TRCs. In some embodiments, asingle target nucleic acid may be detected by a single TRC. In someembodiments, a target nucleic acid may be detected by multiple TRCs,such as multiple replicated TRCs with the same sequence or multiplereplicated TRCs with different sequences. In some embodiments, multipletarget nucleic acids may be detected by a single TRC, multiplereplicated TRCs with the same sequence, or multiple replicated TRCs withdifferent sequences.

In certain embodiments, a target of interest is linear, while in otherembodiments, a target is circular (e.g., plasmid DNA, mitochondrial DNA,or plastid DNA). Target nucleic acid molecules may be modified to primeTRCs for RCR or RCA. Modification methods would create a predictable endsequence on the nucleic acid target molecule by binding and cutting thenucleic acid target at a specific sequence, such as CRISPR/Cas9 orrestriction enzyme digest or by creating a known end using a designedprimer set. Techniques to achieve amenable targets from are performedusing methods known to those skilled in the art.

In general, the pathogenic target sequences can range from a length of10 to 500 nucleotides, 25 to 250 nucleotides, or 50 to 100 nucleotides.The miRNA target sequences may range from a length of 20 to 25nucleotides, such as 21, 22, or 23 nucleotides.

In certain embodiments, a target nucleic acid molecule of interest isabout 100 to about 1,000,000 nucleotides in length. In some embodiments,the target is about 100 to about 1000, about 1000 to about 10,000, about10,000 to about 100,000, or about 100,000 to about 1,000,000 nucleotidesin length. In some embodiments, the target is about 100, about 200,about 300, about 400, about 500, about 600, about 700, about 800, about900, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000,about 6,000, about 7,000, about 8,000, about 9000, about 10,000, about20,000, about 30,000, about 40,000, about 50,000, about 60,000, about70,000, about 80,000, about 90,000, about 100,000, about 200,000, about300,000, about 400,000, about 500,000, about 600,000, about 700,000,about 800,000, about 900,000, or about 1,000,000 nucleotides in length.

In some embodiments, the target nucleic acid(s) is quantified incomparison to control miRNAs. For example, control miRNAs may includemiRNA targets present in the plasma of healthy humans, such as miR-15b,miR-16, miR-24, miR-217, and/or miRNA targets present only in C. eleganswith no sequence similarity to the human genome, such as cel-miR-39,cel-miR-54, and cel-miR-238.

-   -   1. Pathogenic Nucleic Acids

In some embodiments, the target nucleic acid is a nucleic acid thatnaturally occurs in an organism such as a virus, bacteria, or fungus,particularly a pathogenic virus, bacteria, or fungus. A target nucleicacid can be a single-stranded (ss) or double-stranded (ds) nucleic acid.Target nucleic acids can be, for example, DNA, RNA, or the DNA productof RNA subjected to reverse transcription. In some embodiments, a targetmay be a mixture (chimera) of DNA and RNA. In some embodiments, thetarget sequence is complementary to a region of a bacterial, viral, orfungal genome. In some aspects, the pathogenic nucleic acid is a DNA(e.g., cDNA), RNA, or DNA/RNA hybrid with a sequence complementary to asegment of the pathogen genome.

In some embodiments, the viral, bacterial or protozoological targetnucleic acids, are typically selected from viral infectious diseasessuch as influenza, preferably influenza-A, influenza-B, influenza-C orthogotovirus, more preferably influenza-A comprising e.g.,haemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14 or H15, and/or neuroamidase subtypes N1, N2, N3, N4, N5,N6, N7, N8 or N9, or preferably influenza-A subtypes H1N1, H1N2, H2N2,H2N3, H3N1, H3N2, H3N3, H5N1, H5N2, H7N7 or H9N2, etc., or any furthercombination, malaria, severe acute respiratory syndrome (SARS),respiratory syncytial virus infection, yellow fever, AIDS, Lymeborreliosis, Leishmaniasis, anthrax, meningitis, Condyloma acuminata,hollow warts, Dengue fever, three-day fever, Ebola virus, cold, earlysummer meningoencephalitis (FSME), shingles, hepatitis, herpes simplextype I, herpes simplex type II, Herpes zoster, Japanese encephalitis,Arenavirus-associated diseases (Lassa fever infection), Marburg virus,measles, foot-and-mouth disease, mononucleosis infectiosa (Pfeiffer'sglandular fever), mumps, Norwalk virus infection, smallpox, polio(childhood lameness), pseudo-croup, Erythema infectiosum (fifthdisease), rabies, warts, West Nile fever, chickenpox, Cytomegalovirus(CMV); bacterial infectious diseases such as prostate inflammation,anthrax, appendicitis, borreliosis, botulism, Carnphylobacter, Chlamydiatrachomatis (inflammation of the urethra, conjunctivitis), cholera,diphtheria, donavanosis, epiglottitis, typhus fever, gas gangrene,gonorrhoea, rabbit fever, Heliobacter pylori, whooping cough, climaticbubo, osteomyelitis, Legionnaire's disease, leprosy, listeriosis,pneumonia, meningitis, bacterial meningitis, anthrax, otitis media,Mycoplasma hominis, neonatal sepsis (Chorioamnionitis), noma,paratyphus, plague, Reiter's syndrome, Rocky Mountain spotted fever,Paratyphoid fever, Typhoid fever, scarlet fever, syphilis, tetanus,tripper, tsutsugamushi disease, tuberculosis, typhus, vaginitis(colpitis), soft chancre; and infectious diseases caused by parasites,protozoa or fungi, such as amoebiasis, bilharziosis, Chagas disease,Echinococcus, fish tapeworm, fish poisoning (Ciguatera), fox tapeworm,athlete's foot, canine tapeworm, candidosis, yeast fungus spots,scabies, cutaneous Leishmaniosis, lambliasis (giardiasis), lice,malaria, onchocercosis (river blindness), fungal diseases, bovinetapeworm, schistosomiasis, porcine tapeworm, toxoplasmosis,trichomoniasis, trypanosomiasis (sleeping sickness), visceralLeishmaniosis, nappy/diaper dermatitis or miniature tapeworm.

In some aspects, the target nucleic acids are selected from Influenza Avirus, influenza B virus, respiratory syncytial virus, parainfluenzavirus, Streptococcus pneumoniae, Corynebacterium diphtheriae,Clostridium tetani, Measles, Mumps, Rubella, Rabies virus,Staphylococcus aureus, Clostridium difficile, Mycobacteriumtuberculosis, Candida albicans, Haemophilus influenzae B (HiB),poliovirus, hepatitis B virus, human papillomavirus (HPV), humanimmunodeficiency virus, SARS CoV, Pertussis toxin, polio virus,Plasmodium, Staphylococcus aureus, Bordetella pertussis, and/or poliovirus VP1-4. In particular aspects, the viral pathogenic target nucleicacids are specific to human immunodeficiency virus (HIV), herpes simplexvirus (HSV-1), Influenza A virus, West Nile Virus, and/or Epstein-Barrvirus (EBV) viral pathogen nucleic acids.

The following bacteria are of particular interest, because they havebeen found in association with periodontal disease: Actinobacillus(ex.Haemophilus) actinomycetemcomitans; Bacteroides gingivalis; Bacteroidesintermedius Type 1; Bacteroides intermedius Type 2; Eikenella corrodens;Bacteroides forsythus; Fusobacterium nucleatum; Fusobacteriumperio-donticum; Streptococcus intermedius; Wolinella recta. The targetsequences may comprise sequences capable of hybridizing to rRNA of thesebacteria, especially the hypervariable regions of the 16S and 23S rRNA.Other bacterial pathogens include Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus saprophyticus, Streptococcus agalactiae,Streptococcus pyogens, Streptococcus pneumoniae, Enterococcus faecium,Enterococcus faecalis, Mycobacterium tuberculosis, Legionellapneumophilia, Listeria monocytogene, Escherichia coli, Klebsiellapneumoniae, Serratia marcescens, Enterobacter cloacae, Pseudomonasaeruginosa, Stenotrophomonas maltophilia, Proteus mirabilis, Haemophilusinfluenzae, and Neisseria meningitides.

2. miRNAs

In certain embodiments, the target sequence is complementary to a targetmiRNA species. The target miRNA may be a tumor-associated miRNA whichhas elevated levels in a subject with cancer, such as the plasma ofpancreatic cancer patients, or a miRNA which has decreased levels in asubject with cancer. Exemplary pancreatic tumor-associated miRNAsinclude, but are not limited to, miR-let-7, miR-10b, miR-21, miR-25,miR-106b, miR-11, miR-196a, miR-210, miR-212, and miR-221.

The target miRNA can be a miRNA found to be associated with cancersincluding, but not limited to, lung cancer, head and neck cancer, breastcancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer,testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas,pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladdercancer. Further examples cancers include melanomas, malignant melanomas,colon carcinomas, lymphomas, sarcomas, blastomas, renal carcinomas,gastrointestinal tumors, gliomas, prostate tumors, bladder cancer,rectal tumors, stomach cancer, oesophageal cancer, pancreatic cancer,liver cancer, mammary carcinomas, uterine cancer, cervical cancer, acutemyeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloidleukemia (CML), chronic lymphocytic leukaemia (CLL), leukemia,hepatomas, various virus-induced tumors such as, for example, papillomavirus-induced carcinomas (e.g., cervical carcinoma), adenocarcino-mas,herpes virus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced Bcell lymphoma), hepatitis B-induced tumors (hepatocell carcinomas),HTLV-1- and HTLV-2-induced lymphomas, acoustic neuroma, lung carcinomas,small-cell lung carcinomas, pharyngeal cancer, anal carcinoma,glioblastoma, rectal carcinoma, astrocytoma, brain tumors,retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginalcancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome,meningiomas, Schneeberger disease, hypophysis tumor, Mycosis fungoides,carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer,renal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin'slymphomas, urethral cancer, CUP syndrome, head/neck tumors,oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma,oesophageal carcinoma, wart involvement, tumors of the small intestine,craniopharyngeomas, ovarian carcinoma, genital tumors, ovarian cancer,pancreatic carcinoma, endometrial carcinoma, liver metastases, penilecancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytoma, lidtumor, and prostate cancer.

Exemplary human miRNAs associated with bladder cancer includemiR-520c-3p-AS, miR-566-P, miR-33a-AS, miR-1254, miR-487a, miR-1273,miR-541, miR-487, miR-148b, and miR-634. Exemplary human miRNAsassociated with prostate cancer include miR-144*, miR-148a, miR-519b-5p,miR-1324, miR-137, miR-556-5p, miR-330-3p, miR-361-5p, miR-891b,miR-767-5p, miR-744*, miR-208b, miR-548p, miR-20a*, miR-195, miR-33b,miR-1283, miR-519c-5p, miR-497, miR-9*, miR-200a, miR-338-3p,miR-515-5p, miR-31*, miR-551b*, miR-518e*, miR-127-5p, miR-21*,miR-216a, miR-452*, miR-183*, miR-500, miR-1826, miR-625*, miR-513b,miR-526a, miR-33a, miR-1243, miR-517*, miR-541, miR-217, miR-621,miR-518i-5p, miR-873, miR-103-as, miR-450b-5p, miR-545, miR-1251,miR-885-5p, miR-922, miR-628-5p, miR-548f, miR-802, miR-25, miR-423-3p,miR-522*, miR-519a*, miR-455-3p, miR-1245, miR-362-5p, miR-1184,miR-191, miR-487a, miR-216b, miR-525-5p, miR-509-3-5p, and miR-27a*,miR-488*, miR-1226, miR-646, miR-527, miR-635, miR-1825, let-7i*.Exemplary miRNAs associated with breast cancer include miR-1, miR-92a,miR-133a and miR-133b.

Exemplary human miRNAs associated with ovarian cancer include miR-1248,miR-342-3p, miR-133b, miR-605, miR-450b-3p, miR-520a-3p, miR-23b*,miR-423-5p, miR-219-1-3p, miR-454* miR-26b*, miR-1259, miR-655,miR-302c, miR-383, miR-150, miR-412, miR-548i, let-7e*, miR-324-3p,miR-335*, miR-320a, miR-320d, miR-409-3p, miR-590-3p, miR-545*, miR-889,miR-1224-3p, miR-148a*, miR-9, miR-518f, miR-488, miR-182, miR-10a*,miR-19b, miR-15a, miR-1289, miR-500, miR-1281, miR-942, miR-877*,let-7f-1*, miR-651, miR-610, miR-664, miR-613, miR-483-3p, miR-320c,miR-720, miR-299-5p, miR-579, miR-636, miR-197, miR-668, miR-494,miR-1262, miR-578, miR-708*, miR-329, miR-941, miR-155, miR-26a-1*,miR-1246, miR-892b, miR-146a, miR-337-3p, miR-130a*, let-7b, miR-744*,miR-140-3p, miR-573, miR-378, miR-1237, and miR-363*.

Exemplary human miRNAs associated with lung cancer include miR-361-5p,miR-23b, miR-126, miR-527, miR-29a, let-7i, miR-19a, miR-28-5p,miR-185*, miR-23a, miR-1914*, miR-29c, miR-505*, let-7d, miR-378,miR-29b, miR-604, miR-29b, let-7b, miR-299-3p, miR-423-3p, miR-18a*,miR-1909, let-7c, miR-15a, miR-425, miR-93*, miR-665, miR-30e,miR-339-3p, miR-1307, miR-625*, miR-193a-5p, miR-130b, miR-17*,miR-574-5p, and miR-324-3.

In further aspects, the target miRNA may be a diabetes-associated miRNAwhich is associated with diabetes, particular type I Diabetes.Diabetes-associated miRNAs include those miRNAs which are detected ataltered levels in individuals with type I Diabetes and associatedcomplications. For example, miRNAs associated with diabetic retinopathy(e.g., miR146a, miR-195, and miR-320), diabetic nephropathy (e.g.,miR29, miR192, and miR-377), or diabetic neuropathy (e.g., miR-126,miR-203, and miR-503) can be the target miRNA. Further target miRNAsinclude AngiomiRs (e.g., miR-93, miR-200, and miR-661) with implicationsin diabetes as reliable indicators of global microvascular changes.

miRNAs which can be used as control miRNAs for normalization can includemiRNAs present in the plasma of healthy humans (e.g., miR-15b, miR-16,miR-24, and miR-217), and/or miRNAs present in a genome with no sequencesimilarity to the human genome, such as C. elegans (e.g., cel-miR-39,cel-miR-54, and cel-miR-238). Control miRNAs can also compriseartificially-derived sequences such as an ‘alien’ sequence that does notsufficiently align with any known sequences in public genetic databases.

The target miRNA may be a psychiatric disorders-associated miRNA.Psychiatric disorder-associated miRNAs include those miRNAs which aredetected at altered levels in individuals with psychiatric disorders andassociated complications. For example, miRNAs associated with bipolardisorder (e.g., miR-652, miR-132, miR-15b), major depression disorder(e.g., miR-let-7b, miR-let-7c, miR-1202, and miR-135), substance abuse(e.g., miR-124, miR-let-7d, miR-181a, and miR-212), schizophrenia (e.g.,miR-207, miR-298, miR-26b, miR-30b, miR-29b, miR-195, miR-92,miR-30a-5p, miR-30d, miR-20b, miR-29c, miR-29a, miR-212, 106b, miR-7,miR-24, miR-30c, miR-9-3p, miR-let-7g, miR-18b, miR-185, miR-134,miR-674, miR-532, miR-673, miR-224, miR-491, miR-93, miR-383, miR-212,miR-422b, miR-708, miR-540, miR-106b, miR-140, miR-194, miR-325,miR-494, miR-362, miR-409, miR-323, miR-669a, miR-151, miR-18, miR-219,miR-596, miR-597, miR-124-1, miR-598, miR-383,miR-320, miR-486, miR-219,miR-346, and miR-219), and response to anti-depressants (e.g., miR-1202,miR-135, and miR-16) can be the target miRNA.

The target miRNA may be a gynecological disease-associated miRNA.Gynecological disease-associated miRNAs include those miRNAs which aredetected at altered levels in individuals with gynecological disordersand associated complications. For example, miRNAs associated withendometriosis (e.g., miR-451a, miR-20a, miR-29c, miR-145, miR-200a,miR-191, miR-543, miR-141, miR-let-7b, miR-126, miR-17, miR-210,miR-202, miR-122, miR-183, miR-196a, miR-2, miR-22, miR-let-7d, miR-143,miR-195, miR-200b, miR-21, miR-940, miR-4634, miR-100, miR-10b, miR-128,miR-1, miR-215, miR-23a, miR-23b, miR-26a-1, miR-135b, miR-196b,miR-503, miR-504, and miR-629), infertility (e.g., miR-10a, miR-100,miR-184, miR-193a, miR-5p, miR-297, miR-602, miR-625, miR-let-7a/f/d/g,miR-146b, miR-5p, miR-155, miR-182, miR-193a, miR-5p, miR-297, miR-625,miR-602, miR-888, miR-212, miR-662, miR-299-5p, miR-339, miR-5p,miR-20a, miR-486, miR-5p, miR-141, miR-768, miR-5p, miR-376a, miR-15a,miR-21, miR-29a, miR-30d, miR-93*, miR-125-a, miR-320a, miR-7*, miR-425,miR-744, miR-146, miR-7d, miR-202, miR-7e, miR-233b, miR-523a-3p, miR-15a, miR- 188, miR-10a, miR-105, miR-182, miR-372, miR-141, miR-27b,miR-339, miR-3p, miR-345, miR-191, miR-25, miR-302c, miR-196a2,miR-181a, miR-191, miR-372, and miR-645), and polycystic ovariansyndrome (e.g., miR-9, miR-18b, miR-19b, miR-21, miR-27b, miR-30c,miR-93, miR-103, miR-132, miR-135a, miR-146a, miR-155, miR-222, miR-224,miR-320, and miR-383) can be the target miRNA.

The target miRNA may be a neurodegenerative disease-associated miRNA.Neurodegenerative disease-associated miRNAs include those miRNAs whichare detected at altered levels in individuals with neurodegenerativedisease and associated complications. For example, miRNAs associatedwith traumatic brain injury (e.g., miR-195, miR-30d, miR-451, miR-328,miR-92a, miR-486, miR-505, miR-362, miR-151, and miR-20a), Parkinson'sdisease (e.g., miR-let-7, miR-106a*, miR-133b, miR-153, miR-184*,miR-205, miR-21*, miR-224, miR-26b, miR-301b, miR-34b/c, miR-373,miR-433, miR-64, miR-65, miR-7, and miR-9), Tourette's Syndrome (e.g.,miR-429 and miR-106b), Huntington's Disease (e.g., miR-124a, miR-125b,miR-132, miR-146a, miR-150, miR-200a, miR-200c, miR-34b, miR-9, andmiR-9*), Duchenne Muscular Dystrophy (e.g., miR-206, miR-21, miR-31,miR-30c -1, miR-340, miR-208b, miR-499a, miR-4538, miR-4539, miR-208a,miR-95, miR-486-1, miR-539, miR-606, and miR-454), Amyotrophic LateralSclerosis (e.g., miR-124a, miR-146a*, miR-206, miR-338, miR-3p, andmiR-9), Epilepsy (e.g., miR-146a, miR-132, miR-106b, miR-134, miR-155,miR-935, miR-149, miR-196a-2, miR-203a, miR-219a, miR-1, miR-301a,miR-30a, miR-34a, miR-499a, miR-876, miR-1295a, miR-10b, miR-15a,miR-193a, miR-204, miR-21, miR-27a, miR-378a, and miR-487a) andAlzheimer's disease (e.g., miR-106a, miR-106b, miR-107, miR-124a,miR-132, miR-146a, miR-153, miR-181c, miR-29a, miR-29b, miR-29c, andmiR-34a) can be the target miRNA.

The target miRNA may be an autoimmune disease-associated miRNA.Autoimmune disease-associated miRNAs include those miRNAs which aredetected at altered levels in individuals with autoimmune disease andassociated complications. For example, miRNAs associated with rheumatoidarthritis (e.g., miR-146a, miR-155, miR-132 and miR-16), Lupus (e.g.,miR- 101, miR-21, miR-130b, miR- 146a, miR-26a-1, miR-125a, miR-150,miR-638, miR-198, miR-29c, miR-30b, miR-155, miR-371a, miR-422a,miR-423, miR-410, miR-663a, miR-127, miR-221, miR-222 and miR-380),Multiple sclerosis (e.g., miR-429 and miR-106b), Huntington's Disease(e.g., miR-124a, miR-125b, miR-132, miR-146a, miR-150, miR-132, miR-141,miR-17, miR-200a, miR-30a, 572, miR-181c, miR-196a-2, miR-22, miR-27a,miR-499a, miR-1915, miR-106a, 126, miR-214, miR-320a, miR-328, miR-381,miR-422a, miR-20b, miR-633, miR-922, miR-let-7e, miR-let-7g, miR-106b,miR-191, miR-197, miR-199a-2, 19a, miR-19b-1, miR-210, miR-215,miR-219a-1, miR-23b, miR-29b-1, miR-9-2, miR-93, miR-338, miR-372,miR-375, miR-491, miR-146b, miR-614, miR-645 and miR-648), Crohn'sDisease (e.g., miR-99a, miR-375, miR-424, miR-20b, miR-411, miR-629,miR-146a, miR-31, miR-29b-1, miR-122, miR-155, miR-19b1, miR-106b,miR-203a, miR-223, 1246, miR-let-7e, miR-106a, miR-141, miR-191,miR-200b, miR-21, miR-215, miR-29a, miR-320a, miR-595, miR-1286,miR-let-7d, miR-107, miR-124-1, miR-125a, miR-125b-1, miR-126, miR-130a,miR-148a, miR-17, miR-18a, miR-196a-2, miR-19a, miR-20a, miR-200a,miR-200c, miR-206, miR-23a, miR-26b, miR-29c, miR-30-1, miR-9-3,miR-98), and Graft vs Host Disease (e.g., miR-155, miR-586, miR-146a,miR-214, miR-29a, miR-326, miR-199a-2, miR-26b-9, miR-3, miR-374a,miR-377, miR-423, miR-100, miR-200b, miR-30a, miR-489, miR-146b andmiR-411) can be the target miRNA.

The target miRNA may be a cardiovascular disease-associated miRNA.Cardiovascular disease-associated miRNAs include those miRNAs which aredetected at altered levels in individuals with heart disease, heartfailure, and associated complications. For example, miRNAs associatedwith heart failure (e.g., miR-126, miR-210, miR-21, miR-214, miR-30d,miR-150, miR-221, miR-208a, miR-423, miR-499a, miR-208b, miR-145,miR-155, miR-22, miR-25, miR-29a, miR-378a, miR-146a, miR-181c,miR-19b-1, miR-30a, miR-320a, miR-34a, miR-650, miR-665, miR-134,miR-137, miR-182, miR-192, miR-195, miR-199a-2, miR-199b, miR-19a,miR-223, miR-328, miR-377, miR-92b, miR-744, miR-940, miR-1292,miR-1296, miR-1825, miR-1228, miR-1293, miR-663b, miR-3148, miR-3155a,miR-3175, miR-3713, miR-4491, miR-100, miR-107, miR-10b, miR-130b,miR-142, miR-185, miR-206, miR-216a, miR-23a, miR-27b, miR-30b, miR-34b,miR-34c, miR-302c, miR-425, miR-451a, miR-146b, miR-494, miR-518e,miR-568, miR-583, miR-595, miR-29b-1, and miR-340) and heart disease(e.g., miR-21, miR-499a, miR-155, miR-214, miR-126, miR-196a-2,miR-208a, miR-98, miR-221, miR-146a, miR-154, miR-184, miR-181a-1,miR-27b, miR-34b, miR-208b, miR-1265, miR-150, miR-22, miR-29a, miR-34a,miR-10a, miR-145, miR-210, miR-222, miR-378a, miR-421, miR-935,miR-1263, miR-let-7b, miR-let-7c, miR-100, miR-10b, miR-134, miR-142,miR-149, miR-17, miR-186, miR-199b, miR-215, miR-223, miR-23a, miR-24-2,miR-25, miR-26b, miR-29b-1, miR-302a, miR-31, miR-34c, miR-9-3, miR-96,miR-99a, miR-328, miR-377, miR-423, miR-146b, miR-486-1, miR-570 andmiR-650) can be the target miRNA.

3. Additional Targets

TRCs can be used to target other nucleic acids, like ssDNA, dsDNA, orssRNA. In some embodiments, the ssRNA is a ncRNA. In some embodiments,the ncRNA is siRNA, piRNA, miRNA, lncRNA, or snoRNA.

B. Toehold Switch

A high degree of specificity and sensitivity are essential to the TRCsfunctionality. Toehold switches can be used to achieve single miRNAdetection over a wide dynamic range and with exquisite orthogonality.Toehold switches are highly modified versions of bacterialpost-transcriptional riboregulators, which govern translation withrepressive mRNA secondary structure (FIG. 1B). Toehold-mediated stranddisplacement (TMSD) is an enzyme-free entropically-driven process thatfosters invasion of a nucleic acid duplex (i.e., the transducer) by asingle-stranded nucleic acid (i.e., the trigger) based on the invadingstrands ability to gain a “toehold” at its 3′ or 5′-end. The completedisplacement by the trigger may allow a stalled reaction to proceed.Toehold switches display higher hybridization specificity than standardWatson-Crick and padlock based approaches and display greatersingle-nucleotide discrimination.

The toehold switch comprises a first strand with a sequencecomplementary to the target nucleic acid sequence and a second strandwhich forms a double-stranded region or stem switch with part of thefirst strand. The single-stranded region or toehold of the first strandmay comprise a length of 4-20 nucleotides, such as 5, 6, 7, 8, 9, or 10nucleotides in length. The double-stranded region may comprise a lengthof 5-50 nucleotides, particularly 10-20 nucleotides. The toehold regionmay be positioned at the 3′end or the 5′ end of the switch stem (e.g.,is an extension of the 3′end or 5′end of the complement which is part ofthe switch stem). In some embodiments, a toehold region is 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides inlength. In some embodiments, the toehold region is greater than 20nucleotides in length, including for example less than or about 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morenucleotides.

In some embodiments, the TRCs incorporate toehold switches as shortduplex structures and utilize individual miRNAs as triggers. The toeholdforms the double-stranded region under appropriate “hybridizationconditions,” which include temperature, factors such as salts, bufferand pH, detergents, and organic solvents. Finely tuned manipulation ofthe DNA duplex structure with regard to kinetic and thermodynamicproperties alter individual toehold hybridization specificity,effectively creating ON/OFF circuits that may only be triggered by asingle transacting. miRNA species. In some aspects, the 5′-end of aspecific miRNA can bind to the complimentary single-strand sequenceadjacent to the toehold switch and through TMSD, open the duplex. Thehybridization reaction, termed toehold-mediated strand displacement(TMSD), is initiated with the 5′-end binding of the miRNA to a shortsingle-stranded overhang, adjacent to the duplex region of thestructure. Hybridization proceeds with miRNA invasion, internaldisplacement, and duplex uncoupling. An exceptionally high degree ofspecificity is discerned at this step due to the thermodynamicallyfavorable, complete invasion of complimentary miRNA over the unfavorabletransient partial-binding of mismatched, non-targeted miRNA species. Thefully hybridized miRNA is effectively an in situ derived primer thatinitiates rolling circle replication (RCR) of the TRC viahighly-processive, strand-displacing RNA-primed DNA polymerases such asφ29.

Toehold switches dramatically increase the specificity of targetdetection with competitive stand displacement. All other commercialsystems rely on simple complimentary hybridization, which is inherentlyvulnerable to off-target and promiscuous binding, leading tofalse-positives and unreliable data. Embodiments of the presentdisclosure also extend the application of toehold switches bycapitalizing on the exceptional stability of RNA/DNA duplexes andreversing the conventional binding direction in order to initiate DNA,instead of protein, synthesis.

In some aspects, the target nucleic acid is a miRNA of the let-7 familysuch as miR-let-7a and miR-let-7g. Accordingly, in some aspects thetoehold switch sequence is complementary to miR-let-7a(UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 8)) or miR-let-7g(UGAGGUAGUAGUUUGUAUAGUU (SEQ ID NO: 9); which differ by a singlenucleotide at the 12th position (G>U, bolded and underlined), andcomprises the sequence ACTCCATCATCCAACATATCAA (SEQ ID NO: 10) orACTCCATCATCAAACATATCAA (SEQ ID NO:11).

C. Bridge Sequence

In some embodiments, TRCs can contain one or more short,self-complementary sequences, referred to herein as bridge sequences.The inclusion of interspersed, self-complementary bridge sequences candrive intra-molecular hybridization and encourage the formation of thetightly intertwined, but non-entangled nucleic acid sequence structures.Therefore, the product of an individual TRC RCR reaction, referred toherein as a “replicated TRC,” is a singular nucleic acid sequencestructure composed of highly replicated concatenated repeats of the TRCcompliment. The bridge sequences may be incorporated in a variety of TRCconfigurations. In some aspects, the bridge structure is within 1 to 200nucleotides, such as 10 to 100 nucleotides, of the toehold switch. Insome aspects, the bridge structure overlaps with the toehold switch.

The bridge sequence may comprise a secondary structure sequence for theformation of nanospheres from replicated TRCs. This secondary structuresequence or “stabilizing sequence” is a nucleic acid sequence thatfacilitates nanosphere formation and/or stability. For example,palindromic sequences within the bridge sequence can result inhybridization (e.g., intramolecular interactions) between replicatedTRCs resulting in a three dimensional structure of the replicated TRC.These palindromic sequence units can be 5, 6, 7, 8, 9, 10 or morenucleotides in length and of various sequences, such as sequences chosento provide a specific melting temperature. For example, a palindromeAAAAAAATTTTTTT (SEQ ID NO: 12) will provide a 14 bases dsDNA hybridbetween neighboring any two unit replicas in the form of: AAAAAAATTTTTTT(SEQ ID NO: 12) and TTTTTTTAAAAAAA (SEQ ID NO: 13).

The bridge sequence may comprise a low complexity, repetitive sequence.Exemplary low complexity, repetitive sequences may include, but are notlimited to, di-, tri-, and/or tetra-nucleotide repeats (e.g.,GGCCCGGGCCG (SEQ ID NO: 14).

D. Accessory Sequence

In further embodiments, the TRC may comprise accessory sequence(s).Accessory sequences may include secondary structure sequences, sequencescomplementary to capture probe sequences (e.g., for attachment tosurfaces), tagging sequences, sequences for attachment/hybridization oflabel probes, recognition sites for nucleases, such as nicking enzyme,and/or restrictions endonucleases. In some aspects, the accessorysequence of portion thereof (e.g., probe sequence) forms a loop, such aswithin a hairpin or dumbbell structure in which the bridge and/or switchstem regions form the stem portion of the structure.

In certain embodiments, functionally important information can reside inthe accessory sequence(s) of the TRC. Functionally important informationcan include, for example and without limitation, the necessary sequencesfor in vitro replication, unique identification (i.e., barcodes), anddetection. A capture probe recognition sequence may be utilized toimmobilize the TRC and/or replication nanosphere on a substrate, such asan array. An accessory sequence can be used to localize replicatedproducts by hybridization for identification and quantitationapplications. Backbone TRC components are derived from unalignable aliensequences; such sequences have been designed by the NIST External RNAControls Consortium to have zero complementarity within known genomes.

Additional functionality from accessory sequences could include elementsto uniquely digest certain parts of the TRC, TRC-Target hybrid, or TRCproduct for analysis before or after replication. For example, thefollowing sequence has an EcoRV digestion site incorporated so that theproduct can be digested into smaller fragments:

(SEQ ID NO: 15) CAGATATCACcctacctccacatcctccacaagctatccctGATATCtggcggccTCCTCCCGAACTATACAACCTACTACCTCACCGTTGTATAGTT CGCTTCGTGGCCGC.

Additionally, the TRCs could have uracil nucleotides incorporated andcould be substrates for uracil-specific digestion. Accessory sequencesin the replicated TRC could be used to localize replicated TRCs,allowing for detection and analysis without probing but merely buyidentifying where products bind on a microarray. Sequences could be usedto alter the product structure, potentially allowing spatially uniqueproducts for alternative analysis methods.

Additionally, TRCs could have biotin-tagged nucleotides incorporatedthat could be used as anchoring TRCs to a solid support or for isolationof TRCs following construction.

In some embodiments, the accessory sequence comprises one or moreprobing sequences, such as fluorescent probe binding sites. Label probeswill hybridize to the label probe binding sequence and comprise at leastone detectable label. Multiple probing sequences can facilitate codedfluorescent-based digital interrogation and identification of the TRCreaction products. Each TRC may contain a unique set (e.g., combinationand order) of probe sequences. Sequential hybridization with pools ofmulti-state, labeled-probes to the arrayed replicated TRCs followed byimaging can permit decoding, identification, and quantification of themiRNAs present in the sample.

The term “probe” or “probe sequence” is used in a broad sense ofoligonucleotides used in direct hybridization, or as in ligation of twoprobes, or as in probe with an anchor, or as in a probe with an anchorprobe. In some aspects, the probe sequence comprises 5-30 bases,particularly 10-20 bases.

In certain embodiments, the accessory sequence comprises hybridizingsites for binding oligonucleotide probe(s). Certain DNA probes, alsoknown as molecular beacons, are oligonucleotides designed to contain aninternal probe sequence with two ends that are complementary to oneanother. Under appropriate conditions, the ends hybridize togetherforming a stem-loop structure. The probe sequence is contained withinthe loop portion of the molecular beacon and is unrelated to the stemarms. A fluorescent dye is attached to one end on the stem and anon-fluorescent quenching moiety or “quencher” is attached to the otherend of the stem. In the stem-loop configuration, the hybridized armskeep the fluorescent dye and quencher in close proximity, resulting inquenching of the fluorescent dye signal by the well-understood processof fluorescence resonance energy transfer (FRET). When the probesequence within the loop structure finds and hybridizes with itsintended target sequence, the stem structure is broken in favor of thelonger and more stable probe-target duplex. Probe hybridization resultsin the separation of the fluorescent dye and quencher (e.g., the closeproximity is now lost), for which dye can now fluoresce when exposed tothe appropriate excitation source of the detector. Molecular beaconshave been used in a number of molecular biology techniques, such asreal-time PCR, to discriminate allelic differences.

The oligonucleotide probes of the present disclosure can be labeled in avariety of ways, including the direct or indirect attachment ofradioactive moieties, fluorescent moieties, calorimetric moieties, andchemiluminescent moieties. Methodologies for labeling DNA andconstructing DNA adaptors as well as constructing oligonucleotide probesof the present disclosure are known in the art. Many more particularmethodologies applicable to the present methods include linking agents,such as 2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes andtheir phosphoramidite precursors, alkynylamino-nucleotides, nucleotidetriphosphate having a linking group carried on an exocyclic functionalgroup of the base, nucleotide reactive phosphorus derivatives, andfluorescent semiconductor nanocrystals (i.e., quantum dots).

In one aspect, one or more fluorescent dyes are used as labels for theoligonucleotide probes, such as, but not limited to,4,7-dichlorofluoresceins (e.g.,1′,2′,7′,8′-dibenzo-4,7-dichlorofluoresceins), spectrally resolvablerhodamine dyes (e.g., tetramethylrhodamine, rhodamine X, rhodamine 110,and rhodamine 6G); 4,7-dichlororhodamine dyes, ether-substitutedfluorescein dyes (e.g., 2,7-di(aliphatic chalcogen ether substituted) or4,5-di(aliphatic chalcogen ethersubstituted)-9-substituted-6-hydroxy-3H-xanthen-3-one), energy transferdyes, and xanthene dyes (e.g., derivatives of sulfonefluorescein,naphthosulfone fluorescein, fluorescein and naphthofluorescein).

Labeling can also be carried out with quantum dots. The quantum dot maybe a coated nanocrystal capable of light emission includes asubstantially monodisperse core selected from the group consisting ofCdX, where X=S, Se, or Te; and an overcoating of ZnY, where Y=S, Se, andmixtures thereof uniformly deposited thereon, said coated corecharacterized in that when irradiated the particles emit light in anarrow spectral range of no greater than about 40 nm at full width halfmax (FWHM). A nanocrystallite with a diameter of less than 150 Åmay beused, such as a sphere, rod, disk, or other shape. The nanocrystallitecan include a core of a semiconductor material. The core can have anovercoating on a surface of the core. The overcoating can be asemiconductor material having a composition different from thecomposition of the core. Semiconducting nanocrystallites canphotoluminesce and can have high emission quantum efficiencies. Thenanocrystallite can include a core having the formula MX, where M iscadmium, zinc, magnesium, mercury, aluminum, gallium, indium, thallium,or mixtures thereof, and X is oxygen, sulfur, selenium, tellurium,nitrogen, phosphorus, arsenic, antimony, or mixtures thereof. In someembodiments, the nanocrystal may be a luminescent semiconductornanocrystal compound comprising (1) a semiconductor nanocrystal capableof luminescence and/or absorption and/or scattering or diffraction whenexcited by an electromagnetic radiation source (of broad or narrowbandwidth) or a particle beam, and capable of exhibiting a detectablechange in absorption and/or of emitting radiation in a narrow wavelengthband and/or scattering or diffracting when excited; and (2) a linkingagent having a first portion linked to the semiconductor nanocrystal,and a second portion capable of linking to an affinity molecule. In someembodiments, the nanocrystal is a water-soluble nanocrystal that emitsenergy over a narrow range of wavelengths. In some embodiments, thenanocrystal is a semiconductor nanocrystal compound capable of linkingto either one or more second linking agents or to one or more affinitymolecules, and capable of providing a detectable signal in response toexposure to energy.

As used herein, the term “fluorescent signal generating moiety” means asignaling means which conveys information through the fluorescentabsorption and/or emission properties of one or more molecules. Suchfluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, and energy transfer.Commercially available fluorescent nucleotide analogues readilyincorporated into the labeling oligonucleotides or directly into thereplicated TRC include, for example, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP,Cy5-dUTP (Amersham Biosciences, Piscataway, N.J., USA),fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, Texas Red®-5-dUTP,Cascade Blue®-7-dUTP, BODIPY® FL-14-dUTP, BODIPY°R-14-dUTP,BODIPY®TR-14-dUTP, Rhodamine Green™-5-dUTP, Oregon Green® 488-5-dUTP,Texas Red®-12-dUTP, BODIPY® 630/650-14-dUTP, BODIPY® 650/665-14-dUTP,Alexa Fluor® 488-5-dUTP, Alexa Fluor® 532-5-dUTP, Alexa Fluor®568-5-dUTP, Alexa Fluor® 594-5-dUTP, Alexa Fluor® 546-14-dUTP,fluorescein-12-UTP, tetramethylrhodamine-6-UTP, Texas Red®-5-UTP,Cascade Blue®-7-UTP, BODIPY® FL-14-UTP, BODIPY® TMR-14-UTP, BODIPY®TR-14-UTP, Rhodamine Green™-5-UTP, Alexa Fluor® 488-5-UTP, Alexa Fluor®546-14-UTP (Molecular Probes, Inc. Eugene, Oreg., USA). Otherfluorophores available for post-synthetic attachment include, interalia, Alexa Fluor® 350, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor®568, Alexa Fluor® 594, Alexa Fluor® 647, BODIPY 493/503, BODIPY FL,BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568,BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY650/665, Cascade Blue, Cascade Yellow, dansyl, lissamine rhodamine B,Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red(available from Molecular Probes, Inc., Eugene, Oreg., USA), and Cy2,Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, N.J. USA, andothers). FRET tandem fluorophores may also be used, such as PerCP-Cy5.5,PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7; also, PE-Alexa dyes(610, 647, 680) and APC-Alexa dyes. Biotin, or a derivative thereof, mayalso be used as a label on a detection oligonucleotide, and subsequentlybound by a detectably labeled avidin/streptavidin derivative (e.g.,phycoerythrin-conjugated streptavidin), or a detectably labeledanti-biotin antibody. Digoxigenin may be incorporated as a label andsubsequently bound by a detectably labeled anti-digoxigenin antibody(e.g., fluoresceinated anti-digoxigenin). An aminoallyl-dUTP residue maybe incorporated into a detection oligonucleotide and subsequentlycoupled to an N-hydroxy succinimide (NHS) derivitized fluorescent dye,such as those listed supra.

In general, any member of a conjugate pair may be incorporated into adetection oligonucleotide provided that a detectably labeled conjugatepartner can be bound to permit detection. As used herein, the termantibody refers to an antibody molecule of any class, or any subfragmentthereof, such as a Fab. Other suitable labels for detectionoligonucleotides may include fluorescein (FAM), digoxigenin,dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU),hexahistidine (6×His), phosphor-amino acids (e.g., P-tyr, P-ser, P-thr),or any other suitable label. In one embodiment the followinghapten/antibody pairs are used for detection, in which each of theantibodies is derivatized with a detectable label: biotin/α-biotin,digoxigenin/α-digoxigenin, dinitrophenol (DNP)/α-DNP,5-Carboxyfluorescein (FAM)/α-FAM. Probes may also be indirectly labeled,especially with a hapten that is then bound by a capture agent. In onemethod, the method may comprise hybridization with a complementarynucleic acid probe which, via a chemical bond, contains bound at leastone hapten as label which is a steroid bound to at least one position ofthe nucleic acid which does not participate in hydrogen bond formationvia a bridge of at least 4 atoms length, followed by detection of thehybridized probe using labelled anti-hapten antibody. The method maycomprise hybridization with two single-stranded nucleic acid probespresent in the same solution phase complementary to different regions ofthe nucleic acid to be detected, one nucleic acid probe serving asdetector probe and containing as labelling at least one hapten bound viaa chemical linkage and the other nucleic acid probe serving as capturingprobe and being bound to a solid matrix. Many different hapten-captureagent pairs are available for use with the present methods. Exemplaryhaptens include, biotin, des-biotin and other derivatives,dinitrophenol, dansyl, fluorescein, CY5, and other dyes, digoxigenin,digoxigenin derivatives and the like. For biotin, a capture agent may beavidin, streptavidin, or antibodies. Biotin or 2,4-dinitrobenzene may bebonded to a specific oligodeoxyribonucleotide at a specific site otherthan the nucleotide base. Antibodies may be used as capture agents forthe other haptens (many dye-antibody pairs being commercially available,e.g., Molecular Probes).

Probes may be prepared with nucleic acid tag tails instead of beingdirectly labeled. Tails preferably do not interact with test DNA. Thesetails may be prepared from natural bases or modified bases such as isoCand isoG that pair only between themselves. If isoC and isoG nucleotidesare used, the sequences may be separately synthesized with a 5′amino-linker, which allows conjugation to a 5′-carboxy modified linkerthat is synthesized on to each tagged probe. This allows separatelysynthesized tag sequences to be combined with known probes while theyare still attached to the column. In one embodiment, 21 tagged sequencesare used in combination with 1024 known probes.

In some embodiments, in order to distinguish the products of multipletarget miRNA TRCs in multiplex (e.g., 5, 10, 15, 20, or more), twoadditional independent, probing sequences are incorporated into the TRC,each with the potential to bind one of four AlexaFluor labelled beacons.The two-color combination and order of probes binding to any particularnanosphere is unique and indicative of a particular miRNA. Multiplextesting yields highly predictable quantification of each target miRNA.Any presence of unexpected levels of fluorescence would indicatenon-specific TRC/miRNA binding and nanosphere production. The finalmultiplex analysis of all TRCs together is conducted on ordered arraysand utilizes sequential probe hybridization of multi-state, pooledprobes to determine TRC identity by fluorescent imaging.

In certain embodiments, high multiplex ligation assays of probes areused which are not labeled with fluorescent dyes, thus reducingbackground and assay costs. F or example for 8 colors 4×8=32 differentencoding tails may be prepared and 32 probes as a pool may be used inhybridization/ligation. In the decoding process, four cycles each with 8tags are used. Thus, each color is used for 4 tags used in 4 decodingcycles. After each cycle, tags may be removed or dyes photo bleached.The process requires that the last set of probes to be decoded has tostay hybridized through 4 decoding cycles.

In one embodiment, additional properties are included to provide theability to distinguish different probes using the same color, forexample T_(m)/stability, degradability by incorporated uracil bases andUDG enzyme, and chemically or photochemically cleavable bonds. Acombination of two properties, such as temperature stability directly orafter cutting or removing a stabilizer to provide 8 distinct tags forthe same color; more than one cut type may be used to create 3 or moregroups; to execute this 4-8 or 6-12 exposures of the same color may berequired, demanding low photo-bleaching conditions such as low intensitylight illumination that may be detected by intensified CCDs (ICCDs). Forexample if one property is melting temperature (T_(m)) and there are 4tag-oligos or anchors or primers with distinct T_(m), another set of 4oligos can be prepared that has the first 4 probes connected to orintractable with a stabilizer that shifts the T_(m) of these 4 oligosabove the most stable oligo in the first group without stabilizer. Afterresolving 4 oligos from the first group by consecutive melting off, thetemperature may be reduced to the initial low level, the stabilizer maybe cut or removed, and 4 tagged-oligos or anchors or primers can then bedifferentially melted using the same temperature points as for the firstgroup.

II. DETECTION OF TARGET NUCLEIC ACIDS

Certain embodiments of the present disclosure involve the use of TRC(s)provided herein for the detection of target nucleic acids. The TRC(s)may be comprised of DNA, RNA, or analogs thereof, and/or combinationsthereof. In certain embodiments, a TRC comprises one or more non-naturalnucleotides. In some aspects, the target nucleic acids serve as theprimers for rolling circle replication of the TRC to generate a longcontinuous nucleic acid molecule that contains multiple copies of thesame TRC linked in series, referred to herein as concatemers of thereplicated TRCs or replicated nanospheres. The replicated TRCs haveintramolecular hybridization through the bridge sequences (e.g.,stabilizing sequences) to form non-entangled, three-dimensionalreplicated nanospheres. Each nanosphere may comprise at least 100, 200,500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,20,000, 40,000, or 50,000 copies of the TRC depending on the length ofthe RCA reaction and the concentration of the target nucleic acid. Thesenanospheres are then detected using the probe sequences, thus enablingdetection of the target nucleic acids from an initial sample.

A. Target Nucleic Acids

Embodiments of the present disclosure concern compositions and methodsutilizing target nucleic acids from present with or isolated fromsamples. As will be appreciated by those in the art, the sample solutionmay comprise blood, urine, serum, lymph, saliva, anal and vaginalsecretions, perspiration, semen, cells of virtually any organism, withmammalian samples being preferred and human samples being particularlypreferred; environmental samples (including, but not limited to, air,agricultural, water and soil samples); biological warfare agent samples;research samples (e.g., the products of an amplification reaction,including both target and signal amplification, such as PCRamplification reactions; purified samples, such as purified genomic DNA,RNA preparations, raw samples (bacteria, virus, genomic DNA); as will beappreciated by those in the art, virtually any experimental manipulationmay have been done on the samples. In particular embodiments, the targetnucleic acid or population of target nucleic acids can be isolated frombiological samples, such as plasma, saliva, and urine. In someembodiments, the TRC is a “naturally occurring” nucleic acid sequencewhich is a sequence that is present in nucleic acid molecules oforganisms or viruses that exist in nature in the absence of humanintervention. In some embodiments, a target is genomic DNA, messengerRNA, ribosomal RNA, microRNA, pre-microRNA, pro-microRNA, viral DNA,viral RNA, or piwi-RNA.

In some embodiments, the target nucleic acid is a nucleic acid thatnaturally occurs in an organism or a virus. In some embodiments, thetarget nucleic acid is a microRNA. In some embodiments, the targetnucleic acid is present in a plurality of different nucleic acids. Insome embodiments, the target is present as a single copy or in low copy(e.g., less than 0.001%, less than 0.01%, less than 0.1%, or less than1%) in a plurality of different nucleic acids.

B. Rolling Circle Replication

Bacteriophages (or phages), such as ΦX174, M13, lambda, and some virusescan replicate their respective genomes by a “rolling circle” mechanism.An entire genome is reproduced by copying from a circular template.Unlike PCR, the rolling circle mechanism can be performed isothermally(e.g, without the need for heating or cooling cycles). The rollingcircle approach has been used as an in vitro method for replicating(e.g., using one or more primers that copy only original templates) oramplifying (e.g., using two or more primers that copy both originaltemplates as well as copies of templates) nucleic acid molecules ofinterest. For example, circular synthetic oligonucleotide templates,ranging from 34-to-52 bases in size, have been replicated using arolling circle mechanism using E. coli Pol I DNA polymerase and a singleoligonucleotide primer. The rolling circle mechanism using similar sizeconstructs, range 26-to-74, with several polymerases, including E. coliPol I, Klenow DNA polymerase, and T₄ DNA polymerases.

Accordingly, in some aspects, TRCs are replicated, such as to producereplicated TRCs, in a rolling circle replication (RCR) reaction. Conditions and reagents for RCR reactions are known in the art.Generally, RCR reaction components comprise single-stranded DNA circles,one or more primers that anneal to DNA circles (e.g., a target nucleicacid, including pathogenic nucleic acids and miRNAs, can function as insitu derived complementary primers), a DNA polymerase having stranddisplacement activity to extend the 3′ ends of primers annealed to DNAcircles, nucleoside triphosphates, and a conventional polymerasereaction buffer. Such components are combined under conditions thatpermit primers (e.g., target nucleic acids) to anneal to DNA circles andbe extended by the DNA polymerase to form replicated TRCs. An exemplaryRCR reaction protocol is as follows: In a 50 μL reaction mixture, thefollowing ingredients are assembled: 2-50 pmol circular DNA, 0.5units/μL phage φ29 DNA polymerase, 0.2 μg/μL BSA, 3 mM dNTP, 1× φ29 DNApolymerase reaction buffer (Amersham). The RCR reaction is carried outat 30-40° C., particularly 37° C. for 30 minutes to 20 hours,particularly for about 60 minutes. In some embodiments, this and othermethods described herein are performed at a temperature between andincluding room temperature up to and including 50° C., or up to andincluding 40° C., or up to and including 30° C. In some embodiments,this and other methods described herein are performed at about 37° C. Insome embodiments, the concentration of circular DNA in the polymerasereaction may be selected to be low (approximately 10-100 billion circlesper ml, or 10-100 circles per picoliter) to avoid entanglement and otherintermolecular interactions.

Polymerases and reverse transcriptases that are useful in a rollingcircle mechanism generally exhibit the property of strand-displacement,which is the ability to displace a “downstream” nucleic acid strandencountered by the enzyme during nucleic acid synthesis. Thesestrand-displacing enzymes also lack 5′-exonuclease activity. Any stranddisplacing polymerase or reverse transcriptase can be used in rollingcircle replication or rolling circle amplification, for example andwithout limitation, φ29 DNA polymerase, E. coli Pol I, Klenow DNApolymerase, Bst DNA polymerase (large fragment), Bsm DNA polymerase(large fragment), Bsu DNA polymerase (large fragment), Vent(exo-) DNApolymerase, T₇ (exo-) DNA polymerase (T₇ Sequenase), or TopoTaq (achimeric protein of Taq DNA polymerase and topoisomerase V), as well asmutant versions of these DNA polymerases thereof, T₇ RNA polymerase, T₃RNA polymerase, or SP6 RNA polymerase as well as mutant versions ofthese RNA polymerases thereof, or avian myeloblastosis virus reversetranscriptase or Moloney murine leukemia virus reverse transcriptase, aswell as mutant versions of these reverse transcriptases, such asThermoScript reverse transcriptase, Superscript reverse transcriptase orPrimeScript reverse transcriptase. In addition to strand-displacingpolymerases and reverse transcriptases, accessory proteins can furtherenhance the displacement of a downstream nucleic acid strand duringnucleic acid synthesis by increasing the robustness, fidelity, and/orprocessivity of the rolling circle mechanism. Strand-displacingaccessory proteins can be of any type and include, for example andwithout limitation, helicases, single- stranded binding proteins,topoisomerases, reverse gyrases, and other proteins that stimulateaccessory proteins, for example and without limitation, Escherichia coli(E. coli) MutL protein or thioredoxin. DNA helicases are useful in vivoto separate or unwind two complementary or substantially complementaryDNA strands during DNA replication. Helicases can unwind nucleic acidmolecules in both a 5′-to-3′ direction, for example and with limitation,bacteriophage T₇ gene 4 helicase, DnaB helicase and Rho helicase and a3′-to-5′ direction, for example and with limitation, E. coli UvrDhelicase, PcrA, Rep, and NS3 RNA helicase of hepatitis C virus. Helicasemay be obtained from any source and include, for example and withoutlimitation, E. coli helicases (i.e., I, II [UvrD], III, and IV, Rep,DnaB, PriA and PcrA), bacteriophage T₄ gp41, bacteriophage T₇ gene 4helicase, SV40 Large T antigen, Rho helicase, yeast RAD helicase,thermostable UvrD helicases from T. tengcongensis, and NS3 RNA helicaseof hepatitis C virus, as well as mutant versions of these and otherhelicases. Single-stranded binding protein binds single-stranded DNAwith greater affinity that double-stranded DNA. These proteins bindcooperatively, favoring the invasion of single-stranded regions andtherefore destabilizing duplex structures. For example and withoutlimitation, single-stranded binding protein can exhibithelix-destabilizing activity by removing secondary structure and candisplace hybridized nucleic acid molecules. Single-stranded bindingproteins may be obtained from any source and include, for example andwithout limitation, bacteriophage T₄ gene 32 protein, RB 49 gene 32protein, E. coli single-stranded binding protein, cp29 single-strandedbinding protein or bacteriophage T_(7 gene) 2.5, as well as mutantversions of these and other single-stranded binding proteins, such asbacteriophage T₇ gene 2.5 F232L.

The TRCs can be subject to a rolling circle replication using highlyprocessive, strand-displacing polymerases, such as φ29 polymerase. Therolling circle replication can be performed in one or two steps. First,TRCs are allowed to hybridize with complementary primers underappropriate “hybridization conditions,” which include temperature,factors such as salts, buffer and pH, detergents, and organic solvents.Blocking agents such as Bovine Serum Albumin (BSA) or Denhardt's reagentmay be used as part of the hybridization conditions. Second, anappropriate polymerase or replisome and nucleotide mix are provided tothe first reaction mixture to produce amplified or replicated dumbbelltemplates. The hybridization and amplification or replication conditionsare optimized based on several factors, including but not limited, tothe length and sequence composition of the stem region of the dumbbelltemplates, the hybridization conditions, the specific polymerase orreplisome used herein, and the reaction temperature. In certainembodiments, the reaction temperature can be about 10° C. to 65° C. Inother embodiments, the reaction temperature can be about 15° C. to 37°C. In other embodiments, the reaction temperature can be about 20° C. to25° C. In certain embodiments, the temperature is increased in selecttime intervals. For example, without limitations, the reaction ismaintained for five minutes at 10° C., then five minutes at 15° C., fiveminutes at 20° C., then five minutes at 25° C., and five minutes at 30°C.

The RCR and RCA reactions may further comprise a carrier such as abuffer, optionally comprising a preservative, one or more salts, one ormore enzymes such as a polymerase, nucleotides suitable for nucleic acidsynthesis. The reaction may include one or more of the followingreagents: buffer (e.g., KCl, MgCl_(2,) Tris-HCl), dNTPs (e.g., dATP,dCTP, dGTP, dTTP at concentrations of, e.g., about 50 to about 100 μM),polymerase (e.g., at concentrations of about 0.5-2.0 units per 50 μlreaction), and/or water. Salts and buffers include those familiar tothose skilled in the art, including those comprising MgCl₂, and Tris-HCland KCl, respectively. Buffers may contain additives such assurfactants, dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin(BSA) and polyethylene glycol (PEG), as well as others familiar to thoseskilled in the art. Nucleotides are generally deoxyribonucleosidetriphosphates, such as deoxyadenosine triphosphate (dATP), deoxycytidinetriphosphate (dCTP), deoxyguanosine triphosphate (dGTP), anddeoxythymidine triphosphate (dTTP), and are also added to a reactionadequate amount for amplification of the target nucleic acid.

In one exemplary method, based on φ29 polymerase processivity and timetitration data, successful 60 minute reactions produce high-molecularweight (>70 kb) DNA products. Due to the highly entangled nature ofreplicated nanospheres, their migration through agarose gels is markedlydifferent from other oligonucleotides in this size range; this can beconfirmed by gel electrophoresis.

In some embodiments, the target nucleic acid has a length greater thanthat of the TRC to which it hybridizes to, thus producing a 3′ overhangand/or a 5′ overhang. For example, viral nucleic acids from oral samplessuch as saliva, usually contain significantly higher molecular weightfragments (>3 kb). This size discrepancy suggests the presence oflengthy overhanging sequences at both the 5′-end and/or 3′-end ofhybridized pathogenic nucleic acids on TRCs. While the former does notpresent any apparent problem for DNA replication, the presence of 3′-endoverhanging nucleotides does create an impediment. Accordingly, in someaspects, to transform the target nucleic acid into functional primersthe 3′ overhang is removed prior to the RCR reaction. An exonuclease isused to remove the 3′ overhang up to the double-stranded complementaryregion and present a 3′-OH group. In some aspects, the intrinsic3′-exonuclease activity of the DNA polymerase is leveraged to remove the3′-overhang. In other aspects, an exonuclease is introduced such asExonuclease T.

In certain embodiments, exonucleases can be useful to remove undesirednucleic acid molecules that did not successfully ligate to form TRCs.These undesired nucleic acid molecules may have one or more 5′-ends or3′ends that may be in the form of a blunt-ended, 5′-protuding ends,and/or 3′-protruding ends, or may exist in single-stranded form. Theseundesired nucleic acid molecules include, but not limited to,single-stranded nucleic acid molecules, oligonucleotides that may nothave formed into a TRC structure, and unligated hairpin structures.Exonuclease III (also called Exo III) catalyzes the stepwise removal ofmononucleotides from 3′-hydroxyl termini of double-stranded DNA. Alimited number of nucleotides are removed during each binding event,resulting in coordinated progressive deletions within the population ofDNA molecules. The preferred substrates of Exo III are nucleic acidmolecules containing blunt-ends or 5′-protuding ends, although theenzyme also acts at nicks in double-stranded DNA to producesingle-strand gaps. Exo III is not active on single-stranded DNA, andthus 3′-protruding ends are resistant to cleavage. The degree ofresistance depends on the length of the extension, with extensions fourbases or longer being essentially resistant to cleavage. This propertycan be exploited to produce unidirectional deletions from a linearmolecule with one resistant (3′-protruding ends) and one susceptible(blunt-ends or 5′-protruding ends) terminus. Exonuclease III activitydepends partially on helical structure and displays sequence dependence(C>A=T>G). Temperature, salt concentration and the ratio of enzyme toDNA greatly affect enzyme activity, requiring reaction conditions to betailored to specific applications. Exonuclease VII (also called Exo VII)cleaves single-stranded DNA from both 5′→3′ and 3′→5′ direction. Thisenzyme is not active on linear or circular double-stranded DNA. It isuseful for removal of single-stranded oligonucleotide primers andhairpins from a completed PCR reaction and post-ligation reactions whencreating dumbbell constructs. Digestion of single-stranded DNA byExonuclease VII is metal-independent. Exo III and Exo VII can be used incombination to remove undesired nucleic acid molecules that did notsuccessfully ligate to form TRCs.

C. Array

Embodiments of the present disclosure concern detection of thereplicated TRCs. The replicated TRCs can be distributed randomly in anordered pattern on planar or substantially planar slide substrate,fiber-optic substrate, or, semi-conductor device substrates containingwells, depressions, or other containers, vessels, features, orlocations. In some embodiments, the replicated TRCs are dispersed on anarray of optically resolvable discrete spaced apart regions. In someaspects, each discrete spaced apart region has an area of less than one(1) μm², such that substantially all the discrete spaced apart regionshave at most one replicated TRC attached.

The substrate or solid support on which TRCs and primers may be attachedcan be comprised of any material, for example and without limitation, asolid material, a semi-solid material (e.g., [i] a composite of a solidsupport and a gel or matrix material or [ii] linear or cross-linkedpolyacrylamide, cellulose, cross-linked agarose, and polyethyleneglycol), or fluid or liquid material. The substrate can also becomprised of any material that has any dimensions and shape, for exampleand without limitation, square, trapezoidal, spherical, spheroidal,tubular, pellet-shaped, rod-shaped, or octahedral. The substrate shouldcontain properties that are compatible with the present disclosure(e.g., exhibit minimal interference with replication, amplification, ordetection processes). In certain embodiments, the substrate isnonporous. In certain embodiments, the substrate is porous. In certainembodiments, the substrate can be comprised of a hydrophilic porousmatrix, such as a hydrogel. In certain embodiments, the solid materialcomprises, for example and without limitation, a glass material (i.e.,borosilicate, controlled pore glass, fused silica, or germanium-dopedsilica), silicon, zirconia, titanium dioxide, a polymeric material(e.g., polystyrene, cross-linked polystyrene, polyacrylate,polymethylacrylate, polydimethylsiloxane, polyethylene,polyfluoroethylene, polyethyleneoxy, polypropylene, polyacrylamide,polyamide such as nylon, dextran, cross-linked dextran, latex, cyclicolefin polymer, cyclic olefin copolymer, as well as other co-polymersand grafts thereof), or a metallic material. Solid substrates canconsist, for example and without limitation, of one or more membranes,planar surfaces, substantially planar surfaces, non-planar surfaces,microtiter plates, spherical beads, non-spherical beads, fiber-optics,fiber-optics containing spherical beads, fiber-optics containingnon-spherical beads, semi-conductor devices, semi-conductor devicescontaining spherical beads, semi-conductor devices containingnon-spherical beads, slides with one or more wells containing sphericalbeads, slides with one or more wells containing non-spherical beads,filters, test strips, slides, cover slips, or test tubes. In certainembodiments, the semi-solid material comprises, for example and withoutlimitation, linear or cross-linked polyacrylamide, cellulose,cross-linked agarose, and polyethylene glycol.

One or more primers can be attached to a substrate by any suitablemeans. In certain embodiments, the attachment of one or more primers tothe substrate, for example and without limitation, is mediated bycovalent bonding, by hydrogen bonding (i.e., whereby the primer ishybridized with another complementary oligonucleotide covalentlyattached to the substrate and still serves a replication competent oramplification competent function), Van der Waal forces, physicaladsorption, hydrophobic interactions, ionic interactions or affinityinteractions (e.g., binding pairs such as biotin/streptavidin orantigen/antibody). In certain embodiments, one member of the bindingpair is attached to the substrate and the other member of the bindingpair is attached to one or more primers. The attached of one or moreprimers to the substrate occurs through the interaction of the twomember of the binding pair.

Replicated nanospheres may be stochastically loaded onto functionalized,high-density ordered arrays. These arrays, represent an ordered matrixonto which individual replicated nanospheres may discretely andindependently bind. This unbiased but highly organized surface, coupledwith the use of TRC/miRNA-specific fluorescent probing, enables the TRCplatform to efficiently obtain quantitative miRNA population data. Theorganizational capability of the microfabricated array is based onelectrostatic interactions between its dual substrate surface and DNA. Agrid of distinct aminosilane pads induce binding of individualreplicated nanospheres at selective locations by size restriction—only asingle replicated nanosphere can bind at a given aminosilane pad.Hexamethyldisilizane (HMDS) fills the space between the aminosilane padsand discourages promiscuous binding. Importantly, replicated nanospheresare not topologically addressed but may bind indiscriminately to anyunoccupied aminosilane pad.

In some embodiments, fluorescent probes (not beacons) may be used tointerrogate the bound replicated nanospheres at this stage.Single-product identification is based on probe signatures. For example,sixteen different miRNAs can be easily decoded with four colors (i.e.,blue, green, red and yellow) and two probe hybridization events. Morecomplex coding strategies can permit greater flexibility with thepotential to eliminate problematic color combinations. Probing eventsfollow a cyclic pattern of washing, hybridization, washing, and imaging.Following removal of the first probe set, a second probe set ishybridized, washed, and imaged. Identification of fluorescent signaturesis used to decode an individual nanosphere for a particular miRNA.High-resolution imaging is accomplished using an epi-fluorescencedetection microscope. The entire array may be subdivided, and four-colorimages of each subsection are generated corresponding to the specificfluorophores used in decoding. The collated and processed image data isthen decoded and quantified based on the observed probe signatures usingimage analysis software. Using the concentrations of target miRNAs inthe tested populations, an exact target miRNA count can be estimated foreach reaction.

By design, patterned arrays and ordered arrays are expected to providereplicated TRCs template arrays or amplified TRC template arrays thatare spatially and spectrally resolvable for detecting a nucleic acidmolecule. In certain embodiments of a random array, one or more primerscan be covalently bonded to the substrate to form a high-density lawn ofimmobilized primers on a planar or substantially planar surface. The oneor more primers may be attached by any means, for example and withoutlimitation, by methods involving dropping, spraying, plating orspreading a solution, emulsion, aerosol, vapor, or dry preparation. Byintroducing dumbbell templates onto the substrate in limiting dilutionfashion, one or more primers will contact the dumbbell template,enabling the rolling circle mechanism in the presence of polymerase toproduce one or more replicated dumbbell templates (e.g.., replicateddumbbell template array) or amplified dumbbell templates (e.g.,amplified dumbbell template array) that are spatially and spectrallyresolvable for detecting a nucleic acid molecule. In certain embodimentsof a random assortment in patterned arrays, one or more primers can becovalently bonded to the substrate to form high-density, immobilizedprimers on one of more spherical or non-spherical beads. By introducingdumbbell templates onto the substrate in limiting dilution using an oilin water emulsion system, one or more primers will contact the dumbbelltemplate, enabling the rolling circle mechanism to produce one or morereplicated dumbbell templates or amplified dumbbell templates. Incertain embodiments, replicated dumbbell templated beads or amplifieddumbbell templated beads can be enriched to remove those beads thatfailed to replicate or amplify dumbbell templates based on Poissonstatistics of distributing single molecules. Replicated dumbbelltemplated beads or amplified dumbbell templated beads, with or withoutenrichment, can then be distributed randomly in a ordered pattern onplanar or substantially planar slide substrate, fiber-optic substrate,or, semi-conductor device substrates containing wells, depressions, orother containers, vessels, features, or locations. In other certainembodiments of a random assortment in patterned arrays, one or moreprefabricated hydrophilic features (i.e., spots) on the surface can besurrounded by hydrophobic surfaces for the covalent bonding of one ormore primers to the substrate. For example and without limitation,patterned arrays can be created photolithographically etched, surfacemodified silicon substrates with grid-patterned rrays of ˜300 nm spots.By introducing the dumbbell templates onto a patterned substrate inlimiting dilution fashion, the primer will contact the dumbbelltemplate, enabling the rolling circle mechanism to produce one or morereplicated dumbbell templates or amplified dumbbell templates. Incertain embodiments, the prefabricated hydrophilic spots can be madesmall even to accommodate only one replicated dumbbell template oramplified dumbbell template. As distributing single molecules based onPoisson statistics results in a considerable fraction of no templatespots, following the rolling circle procedure, additional rounds ofdistributing, contacting, and rolling circle may be employed to increasethe density of replicated dumbbell templates or amplified dumbbelltemplates on the substrate. In certain embodiments of “knowns” patternedin ordered arrays, one or more known primers can be printed (i.e.,spotted arrays) or made in situ at addressable locations on thesubstrate. By introducing the dumbbell templates onto a patternedsubstrate in limiting dilution fashion, one or more primers will contactthe dumbbell template, enabling the rolling circle mechanism to produceone or more replicated dumbbell templates or amplified dumbbelltemplates.

Accordingly, in some embodiments the replicated TRCs can bestochastically loaded onto functionalized, high-density ordered arrays.The arrays can be photolithographically etched, surface-modified (SOM)silicon substrates with grid-patterned arrays. The individual replicatedTRCs can discretely and independently bind the array. This unbiased buthighly organized surface, coupled with the use of TRC/target nucleicacid-specific probing, enables the efficient detection andquantification of the target nucleic acid population. The organizationalcapability of the microfabricated array is based on electrostaticinteractions between its dual substrate surface and DNA. A grid ofdistinct aminosilane pads induce binding of individual replicated TRCsat selective locations by size restriction—only a single replicated TRCscan bind at a given aminosilane pad. Hexamethyldisilizane (HMDS) fillsthe space between the aminosilane pads and discourages promiscuousbinding. The replicated TRCs will not be topologically addressed but maybind indiscriminately to any unoccupied aminosilane pad.

Generally, densities of single molecules are selected that permit atleast 20%, or at least 30%, or at least 40%, or at least a majority ofthe molecules to be resolved individually by the signal generation anddetection systems used. Whenever optical microscopy is employed, forexample with molecule-specific probes having fluorescent labels, adensity is selected such that at least a majority of single moleculeshave a nearest neighbor distance of 200 nm or greater; and in anotheraspect, such density is selected to ensure that at least seventy percentof single molecules have a nearest neighbor distance of 200 nm orgreater. In still another aspect, whenever optical microscopy isemployed, for example with molecule-specific probes having fluorescentlabels, a density is selected such that at least a majority of singlemolecules have a nearest neighbor distance of 300 nm or greater; and inanother aspect, such density is selected to ensure that at least seventypercent of single molecules have a nearest neighbor distance of 300 nmor greater, or 400 nm or greater, or 500 nm or greater, or 600 nm orgreater, or 700 nm or greater, or 800 nm or greater. In still anotherembodiment, whenever optical microscopy is used, a density is selectedsuch that at least a majority of single molecules have a nearestneighbor distance of at least twice the minimal feature resolution powerof the microscope. In another aspect, polymer molecules of the presentdisclosure are disposed on a surface so that the density of separatelydetectable polymer molecules is at least 1000 per μm², or at least10,000 per μm², or at least 100,000 per μm².

In another aspect of the disclosure, the requirement of selectingdensities of randomly disposed single molecules to ensure desirednearest neighbor distances is obviated by providing on a surfacediscrete spaced apart regions that are substantially the sole sites forattaching single molecules. That is, in such embodiments the regions onthe surface between the discrete spaced apart regions, referred toherein as “inter-regional areas,” are inert in the sense that thereplicated TRCs do not bind to such regions. In some embodiments, suchinter-regional areas may be treated with blocking agents, e.g., DNAsunrelated to replicated TRC DNA, other polymers, and the like.Generally, the area of discrete spaced apart regions is selected, alongwith attachment chemistries, macromolecular structures employed, and thelike, to correspond to the size of single molecules of the presentdisclosure so that when single molecules are applied to surfacesubstantially every region is occupied by no more than one singlemolecule. The likelihood of having only one single molecule per discretespaced apart region may be increased by selecting a density of reactivefunctionalities or capture oligonucleotides that results in fewer suchmoieties than their respective complements on single molecules. Thus, asingle molecule will “occupy” all linkages to the surface at aparticular discrete spaced apart region, thereby reducing the chancethat a second single molecule will also bind to the same region. Inparticular, in one embodiment, substantially all the captureoligonucleotides in a discrete spaced apart region hybridize to adaptoroligonucleotides a single macromolecular structure. In one aspect, adiscrete spaced apart region contains a number of reactivefunctionalities or capture oligonucleotides that is from about tenpercent to about fifty percent of the number of complementaryfunctionalities or adaptor oligonucleotides of a single molecule. Thelength and sequence(s) of capture oligonucleotides may vary widely, andmay be selected in accordance with well-known principles.

In one aspect, the lengths of capture oligonucleotides are in a range offrom 6 to 30 nucleotides, and in another aspect, within a range of from8 to 30 nucleotides, or from 10 to 24 nucleotides. Lengths and sequencesof capture oligonucleotides are selected (i) to provide effectivebinding of macromolecular structures to a surface, so that losses ofmacromolecular structures are minimized during steps of analyticaloperations, such as washing, etc., and (ii) to avoid interference withanalytical operations on analyte molecules, particularly when analytemolecules are DNA fragments in a nanosphere. In regard to (i), in oneaspect, sequences and lengths are selected to provide duplexes betweencapture oligonucleotides and their complements that are sufficientlystable so that they do not dissociate in a stringent wash. In regard to(ii), if DNA fragments are from a particular species of organism, thendatabases, when available, may be used to screen potential capturesequences that may form spurious or undesired hybrids with DNAfragments. Other factors in selecting sequences for captureoligonucleotides are similar to those considered in selecting primers,hybridization probes, oligonucleotide tags, and the like.

In one aspect, the area of discrete spaced apart regions is less than 1μm²; and in another aspect, the area of discrete spaced apart regions isin the range of from 0.04 μm² to 1 μm²; and in still another aspect, thearea of discrete spaced apart regions is in the range of from 0.2 μm² to1 μm². In another aspect, when discrete spaced apart regions areapproximately circular or square in shape so that their sizes can beindicated by a single linear dimension, the size of such regions are inthe range of from 125 nm to 250 nm, or in the range of from 200 nm to500 nm. In one aspect, center-to-center distances of nearest neighborsof such regions are in the range of from 0.25 μm to 20 μm; and inanother aspect, such distances are in the range of from 1 μm to 10 μm,or in the range from 50 to 1000 nm. In particular aspects, spaced apartregions for immobilizing nanospheres are arranged in a rectilinear orhexagonal pattern.

In certain embodiments of the disclosure, photolithography, electronbeam lithography, nano imprint lithography, and nano printing may beused to generate such patterns on a wide variety of surfaces. Thesetechniques can be used to generate patterns of features on the order of1/10^(th) of a micron and have been developed for use in thesemiconductor industry. In one embodiment, a single “masking” operationis performed on the DNA array substrate, as opposed to the 20 to 30masking operations typically needed to create even a simplesemiconductor. Using a single masking operation eliminates the need forthe accurate alignment of many masks to the same substrate. There isalso no need for doping of materials. Minor defects in the pattern mayhave little to no effect on the usability of the array, thus allowingproduction yields to approach 100%. In certain embodiments,stereolithography (i.e., sterolithography 3D printer or digital lightprocessing 3D printer) can be used instead of lithography techniques toprint the array device.

In one embodiment, high density structured random DNA array chips havecapture oligonucleotides concentrated in small, segregated capture cellsaligned into a rectangular grid formation. Preferably, each capture cellor binding site is surrounded by an inert surface and may have asufficient but limited number of capture molecules (100-400). Eachcapture molecule may bind one copy of the matching adaptor sequence onthe RCR produced nanosphere. By providing enough RCR products almostevery spot on the array may contain one and only one unique TRC target.

In one exemplary method, the replicated TRCs can be pipettedindividually on to separate ordered arrays. After a short incubationperiod which allows the replicated TRCs to bind to the functionalizedsurface, the arrays are then prepared for probe hybridization by rinsingin a neutral pH buffer to remove contaminants from the initial reaction.

D. Probe Detection

The oligonucleotide product of an individual TRC reaction (one targetoligonucleotide hybridizing with a single TRC) is composed ofconcatenated replicates of the TRC compliment sequence. Depending on thereaction conditions (duration and polymerase), the overall length may betens of kilobases and the number of replicates can range from tens tothousands. The product oligonucleotide forms a tertiary structure withboth single and double-stranded domains. The rapid replication processand lengthy product can be detected by non-specific methods or moredirected sequence-specific detection methods may be employed.

In certain embodiments, the replicated TRCs can be detected witholigonucleotide probes. The oligonucleotide probes can be labeledoligonucleotide probes. The oligonucleotide probes can be labeled DNAprobes. In certain embodiments, the oligonucleotide probe can beattached to one or more of a fluorophore, a chromophore, a radioisotope,an enzyme, or a luminescent compound, or combinations thereof.

In certain embodiments, the replicated TRCs can be detected with a rangeof instruments capable of detecting various accessory sequences, probesand moieties within the replicated TRC. Exemplary instruments include a(FIG. 2C) machine for detection of fluorescent molecular probes or Sybrstaining of replicated products in real-time; a fluorescent orcolorimetric plate reader for detection of replicated TRCs byfluorescent dyes, probes or pH sensitive stains at the completion of RCRor RCA; a scanning fluorescence microscope for the detection andquantitation of replicated TRCs on stochastically arranged or locationspecific arrays; imaging using smartphones or other portable handheldimaging devices for use with colorimetric or photosensitive probes ordyes; flow cytometers capable of detecting fluorescent probes bound toreplicated TRCs or fluorescent nucleotides incorporated into replicatedTRCs.

Automation of the probe hybridization reactions and image collection maybe accomplished with standard microfluidic devices and modifications toa microscope. Briefly, the former includes the use ofcomputer-controlled microfluidic valves and peristaltic pumps tosequentially deliver reagents for washing and probe hybridization. Thecoordinated, intermittent four-color imaging is accomplished using ascanning XY-stage and the Nikon Perfect Focus System, which minimizesbackground and image drift.

1. Non-Specific Detection Methods

TRC products can be detected by single or double-strandedoligonucleotide intercalating dyes (stains) (Sybr, EtBr, etc.). The dyesbind non-specifically to oligonucleotides in various forms and fluoresceunder the proper excitation wavelengths, indicating both a positivereaction and with the correct instruments, a relative quantity ofproduct that can indicate the amount of starting material.

In this method, a positive reaction between TRC and targetoligonucleotide would produce a product that could be detected with Sybrin real-time using an RT-PCR instrument or as an end-point detectionmethod using a microplate reader. Using a titration scale, the productscould be quantified and equated to the starting target concentration. Ina simplified version, the TRC reaction could be read out simply as apositive or a negative for the target oligonucleotide.

In a similar manner, a positive reaction between TRC and targetoligonucleotide could be detected with a colorimetric dye, many of whichare pH sensitive. The rapid TRC replication reaction would reduce pH,producing a color change detectable with a microplate reader,spectrophotometer, or by the naked eye. The reaction could be identifiedas positive or negative, or the initial target oligonucleotideconcentration could be calculated.

An additional method of non-specific detection involves theincorporation of modified nucleotides into the product. These couldinclude fluorescently labeled, radiolabeled, or biotinylatednucleotides. The isolated products could then be quantified withappropriate detection instruments.

2. Sequence Specific Detection Methods

TRCs may contain sequence elements that correspond with a unique target.The resulting target-specific TRC reaction products have highlyreplicated domains that permit sequence-specific product isolation,detection, and/or quantification, notably for use in multiplex reactionsystems. Multiple such sequences may be incorporated to achieve higherorder multiplexing, possibly requiring multiple probing cycles.

Engineered sequence domains that are highly replicated during the TRCreaction may be bound with fluorescent oligonucleotides in the form ofprobes or beacons. Probes are short sequences with attached fluorophoresthat hybridize with their complimentary sequence; beacons are similar,but contain a cis-acting quencher to mask the fluorescent signal whenbeacons are not bound to the target sequence. Following hybridization,excess probe or beacon is washed away and analysis of the bound productsmay proceed with overall fluorescence, or single molecule quantificationand decoding.

For example, four target-specific TRCs may be used to analyze fourdifferent target concentrations in a single reaction. Four beacons withfour different fluorescent signals are used. During the reaction, asingle target-specific beacon binds to TRC reaction productcorresponding with that target oligonucleotide. The level offluorescence can be monitored during the reaction or at end-point.Similarly, the level of fluorescence for the other three targets mayalso be monitored in the same way.

Detection methods may combine various methods—for example, TRC reactionscould be run with labeled nucleotides; the products of which may then bedistributed on a location specific array to be quantified by scanningfluorescent microscopy.

Alternatively, the highly-entangled but discrete products of the TRCreaction may be distributed on an ordered array based on variouschemistries. Probe or beacon hybridization can take place prior to orafter arraying. Individual product detection and quantification can thenproceed using a microarray reader or fluorescence microscopy.

An alternative sequence-specific detection method involves isolation ofa TRC reaction product to a specific, known location on an orderedarray. Hybridization of the product is achieved with immobilizedoligonucleotides that are specific to the target product; TRC reactionproducts would then be captured at a defined location for quantificationbased on array address. In this manner, multiple targets could beanalyzed in the same reaction on the same slide.

Single-product identification can be performed. For example, sixteendifferent miRNAs can be easily decoded with four colors (e.g., blue,green, red and yellow) and two probe hybridization events. More complexcoding strategies will permit greater flexibility with the potential toeliminate problematic color combinations. Probing events follow a cyclicpattern of washing, hybridization, washing, and imaging. Followingremoval of the first probe set, a second probe set will be hybridized,washed, and imaged. Identification of fluorescent signatures can be usedto decode an individual nanosphere for a particular miRNA.High-resolution imaging is accomplished using an epi-fluorescencedetection microscope. The entire array is subdivided, and four-colorimages of each subsection is generated corresponding to the specificfluorophores used in decoding. The collated and processed image data isthen be decoded and quantified based on the observed probe signatures.Coordinated, intermittent four-color imaging can be accomplished using ascanning XY-stage and Nikon Perfect Focus system, which minimizesbackground and image drift. The digital readout displays tightcorrelation (e.g., <5% from calculated count) with predicted speciescounts for the constructed target populations with sensitivity (e.g.,<10 fM) and specificity (e.g., DF>30).

Using the concentrations of target miRNAs in the tested populations, anexact target miRNA count can be estimated for each reaction. The numberof miRNA-specific nanospheres detected on the array should besignificantly close to this value. Fluorescence intensity may be used toindicate replicated TRC size.

Signals from replicated TRCs on arrays can generated and detected by anumber of detection systems, including, but not limited to, scanningelectron microscopy, near field scanning optical microscopy (NSOM), andtotal internal reflection fluorescence microscopy (TIRFM). Theapplication of such techniques for analyzing and detecting nanoscalestructures on surfaces is known in the art.

In one aspect, instruments for use with arrays of the present disclosurecomprise three basic components: (i) a fluidics system for storing andtransferring detection and processing reagents, e.g., probes, washsolutions, and the like, to an array; (ii) a reaction chamber, or flowcell, holding or comprising an array and having flow-through andtemperature control capability; and (iii) an illumination and detectionsystem. In one embodiment, a flow cell has a temperature controlsubsystem with ability to maintain temperature in the range from about5-95° C., or more specifically 10-85° C., and can change temperaturewith a rate of about 0.5-2° C. per second.

In one embodiment, four or more cameras may be used, preferably in the10-16 megapixel range. Multiple band pass filters and dichroic mirrorsmay also be used to collect pixel data across up to four or moreemission spectra. To compensate for the lower light collecting power ofthe decreased magnification objective, the power of the excitation lightsource can be increased. Throughput can be increased by using one ormore flow chambers with each camera, so that the imaging system is notidle while the samples are being hybridized/reacted. Because the probingof arrays can be non-sequential, more than one imaging system can beused to collect data from a set of arrays, further decreasing assaytime.

In one aspect, suitable illumination and detection system forfluorescence-based signal is a Zeiss Axiovert 200 equipped with a TIRFslider coupled to an 80 milliwatt 532 nm solid state laser. The sliderilluminates the substrate through the objective at the correct TIRFillumination angle. TIRF can also be accomplished without the use of theobjective by illuminating the substrate though a prism optically coupledto the substrate. Planar wave guides can also be used to implement TIRFon the substrate Epi illumination can also be employed. The light sourcecan be rastered, spread beam, coherent, incoherent, and originate from asingle or multi-spectrum source.

3. Bead-Based Detection

TRC technology may be used for the identification of concrete miRNAdisease signatures and for efficiently and effectively analyzing miRNApopulations in patients, providing comprehensive health information in aconvenient and functional platform. In some embodiment, integration ofthe TRC technology with the medium-plex Luminex 200 platform (e.g., upto 100 assays) delivers a robust, scalable, and cost-effective solutionto replace underperforming protein, DNA, and RNA-based tests.

Typical amplification-based protocols demand that samples be dividedover multiple reactions, with each reaction targeting a separate anddistinct miRNA. This can be problematic given the already lowconcentration of circulating miRNAs and the small volume of preciousclinical samples. More detrimental is the likelihood that rare butinformative miRNA species will be diluted out over many misguidedreactions, leading to false negatives. The streamlined TRC approach usesa simplified reaction in which all target miRNAs are bound andreplicated in a single tube, eliminating the possibility of dilutionbias and making subtle variations in target miRNA easier to detect overbackground noise.

Luminex xMAP microspheres incorporate a number of features that can becomplimentary to TRC miRNA detection and an ideal platform fordiagnostic assay development. (i) Oligonucleotides may be covalentlycoupled to surface of each microsphere in varying densities. (ii)Microspheres are embedded with preselected ratios of two or threefluorescent dyes, enabling deconvolution of each reaction target uponmicrosphere detection. Upon target assignment, the platform analyzerfurther detects the presence of fluorescent reporter molecules bound tothe surface of each bead. (iii) MagPlex microspheres are magnetized tofacilitate rapid isolation.

In some embodiments, each bead-type contains a specific TRC constructbound to the surface along with cis-TRC-specific secondary primers.Specific bead types indicate the target miRNA species and the presenceof a fluorescent signal, incorporated as a labeled nucleotide during RCRand subsequent rolling circle amplification (RCA), indicate a positivemiRNA-TRC reaction. Functionalized, product-bound beads are isolated forrapid production and analysis on the Luminex platform.

Luminex MagPlex beads contain carboxyl groups to facilitate theattachment of numerous macromolecules, including nucleic acids. As TRCsare circular in nature, amino-modifier C6 dT phosphoramidates may beused during synthesis of TRC oligonucleotides for coupling to Luminexbeads. TRC-specific, complementary, secondary primers may also beattached to the Luminex beads to further extend the unique replicatedTRC products, covalently attaching such products to the beads. Thisprocess results in a highly-intertwined but discrete, bead-productdetectable on the Luminex 200 platform (FIG. 6).

To label replicated TRC products for detection on Luminex platforms,fluorescently-labelled nucleotides (e.g., Fluorescein-12-dCTP;PerkinElmer) may be utilized. Previous experience with Bst and Bsupolymerases have demonstrated efficient incorporation of such modifiednucleotides in RCR. Alternatively, an intercalating dye such as Sybrcould be used to detect replicated TRCs bound to beads.

Microsphere analysis may be carried out according to the standardoperating protocol provided by Luminex. The Luminex 200 instrument iscapable of simultaneously analyzing up to 100 unique reactions (e.g.,100 miRNA targets) with more advanced versions of the technology capableof handling up to 500 unique reactions. Luminex' s xPONENT software maybe used for continuous monitoring and quantification of all targetmiRNAs.

III. METHODS OF USEe

In some embodiments, the methods provided herein concern the utilizationof pathogenic nucleic acid or miRNA signatures as reliable, circulatingbiomarkers for disease detection, stratification, and intervention. Insome aspects, the methods are used for the early detection of cancer,particularly pancreatic cancer. The dynamic, disease-specific, andintervention-responsive nature of extra-cellular and circulating miRNApopulations is used to indicate disease pathogenesis and progression.Because of the quantitative nature of detection methods, thecompositions and methods described herein can be used to quantitatetarget molecules whose abundance is indicative of a biological state ordisease condition, for example, blood markers that are upregulated ordownregulated as a result of a disease state. In addition, thecompositions and methods described herein can be used to provideprognostic information that assists in determining a course of treatmentfor a patient. For example, the amount of a particular marker, such asmiRNA, for a tumor can be accurately quantified from even a small samplefrom a patient.

In certain embodiments, the TRC platform of the present disclosure isused for the detection, stratification, and intervention of conditionsincluding various infectious diseases such as those caused by viruses,e.g., HIV-1, EBV, hepatitis, herpes viruses, enteric viruses,respiratory viruses, rhabdovirus, rubeola, poxvirus, paramyxovirus,morbillivirus, etc. are of interest. Infectious agents of interest alsoinclude bacteria, such as Pneumococcus, Staphylococcus, Bacillus,Streptococcus, Meningococcus, Gonococcus, Escherichia, Klebsiella,Proteus, Pseudomonas, Salmonella, Shigella, Hemophilus, Yersinia,Listeria, Corynebacterium, Vibrio, Clostridia, Chlamydia trachomatis,Mycobacterium, Helicobacter and Treponema; protozoan pathogens, and thelike.

The methods of the present disclosure are suitable for diagnosing anydiseases for which a differential expression of miRNAs compared tohealthy controls or other diseases exists. In particular, the method maybe used for diagnosing cancer including bladder cancer, brain cancer,breast cancer, colon cancer, endometrium cancer, gastrointestinalstromal cancer, glioma, head- and neck cancer, kidney cancer, leukemia,liver cancer, lung cancer, lymph node cancer, melanoma, meninges cancer,ovarian cancer, pancreas cancer, prostate cancer, sarcoma, stomachcancer, testicular cancer, thyroid cancer, thymus cancer and Wilms'tumor. The diagnosis may comprise determining type, rate and/or stage ofcancer. The course of the disease and the success of therapy such aschemotherapy may be monitored. The method of the present disclosureprovides a prognosis on the survivor rate and enables to determine apatient's response to drugs.

The present methods may also be used for diagnosing chronic obstructivepulmonary disease (COPD), endometriosis, polycystic ovarian syndrome,multiple sclerosis, lupus, liver disease, Parkinson's disease,Alzheimer's disease, arthritis, gout, glucose metabolism disorders,alcoholism, lipid metabolism disorders, retinal and other oculardisorders and diseases, emphysema, pelvic organ prolapse,substance-induced psychoses, esophagitis, aortic diseases, placentaprevia, stomach ulcers, coronary restenosis, irritable bowel syndrome,metabolic bone diseases, hemophilia, cardiomyopathies, insulinresistance, cardiotoxicity, hypertension, coronary artery disease,Aicardi-Goutières syndrome, glaucoma, intestinal diseases, kidneyneoplasms, prostatitis, benign prostatic hypertrophy, hormonal disordersand embolisms.

In certain embodiments, the TRC platform is used for the detection andmonitoring of type I Diabetes. The timely diagnosis and management ofmicrovascular complications in type 1 Diabetes (T1D) represents achallenging paradigm for clinicians and a pressing need for thosesuffering from the disease. Current diagnostic approaches fail in theirability to sensitively, specifically, and comprehensively detect theearliest molecular changes that underlie such complications, nor do theyhave the ability to resolve individual complication risk and progressionat the earliest stages, when intervention is most effective.Accordingly, embodiments of the present disclosure provide a noveldetection and surveillance platform for the digital quantitation ofcell-free miRNA signatures indicative of risk and progression for threeimportant T1D microvascular complications: diabetic retinopathy (DR),diabetic nephropathy (DN), and diabetic peripheral neuropathy (DPN).Clinical intervention, when initiated in the early stages of thesecomplications, can limit their progression, preventing vision loss,preserving kidney function, minimizing neurological pain and reducingthe risk of limb loss, potentially saving billions of dollars andpreserving quality of life in these patients. Current methods fordiagnosing, stratifying, and monitoring T1D complications rely on amuddled collection of biomarker analyses, invasive procedures andphysical exams. They have significant disadvantages that limit theirability to efficiently and effectively meet the demands of a growing,diverse diabetic population. These methods are expensive,labor-intensive, and time-consuming. For example, DR screening dependson the observation of altered ocular vascular morphology andnecessitates costly imaging platforms, image processing equipment anddigital image archiving, along with the need for highly skilledpractitioners to operate the technology and interpret such results. DNrelies on imprecise urine microalbumin testing or invasive kidneybiopsy, and DPN is most frequently diagnosed following physical exam bya physician after patient discomfort or ulcer formation. The presenttechnology can be used to screen a curated library of miRNAs shown to beinvolved in T1D DR and metabolic regulation in both surrogate controlbiofluids and clinical patient samples. In using clinical patientsamples, the miRNA expression profiles can be correlated to T1D DRprogression and characterize molecular subtypes with therapeutic value.Leveraging these early complication-specific miRNA signatures, theadvanced diagnostic technology can provide fundamental and timelyinformation to positively affect diagnosis and management of T1D DR,preserving quality of life for those suffering. This technologyrepresents a broadly enabling approach, capable of providingcomprehensive diabetic health information in a convenient and accessibleplatform.

In certain embodiments, the TRC platform is used for the detection andmonitoring of psychiatric disorders. Utilizing miRNA as biomarkers forbipolar disorder, schizophrenia, major depressive disorder, substanceabuse, and predicting responses to antidepressants can identify andstratify these disorders leading to more productive pharmaceuticalintervention strategies.

In certain embodiments, the TRC platform is used for the detection andmonitoring of gynecological diseases. Utilizing miRNA as biomarkers forendometriosis, infertility, and polycystic ovarian syndrome can identifyand stratify these disorders leading to more productive pharmaceuticalintervention strategies.

In certain embodiments, the TRC platform is used for the detection andmonitoring of neurodegenerative diseases. Utilizing miRNA as biomarkersfor traumatic brain injury, Parkinson's disease, Alzheimer's disease,Tourette's syndrome, Huntington's disease, Duchenne Muscular Dystrophy,Amyotrophic Lateral Sclerosis, and epilepsy can identify and stratifythese disorders leading to more productive pharmaceutical interventionstrategies.

In certain embodiments, the TRC platform is used for the detection andmonitoring of autoimmune diseases. Utilizing miRNA as biomarkers forrheumatoid arthritis, lupus, multiple sclerosis, Crohn' s disease, andgraft vs host disease can identify and stratify these disorders leadingto more product pharmaceutical intervention strategies.

In certain embodiments, the TRC platform is used for the detection andmonitoring of cardiovascular diseases. Utilizing miRNA as biomarkers forhearts failure and heart disease can identify and stratify thesedisorders leading to more product pharmaceutical interventionstrategies.

In certain embodiments, a TRC-based approach is used to diagnose aparticular disease. In certain embodiments, a TRC-based approach is usedto diagnose, monitor, and stratify a particular disease. In certainembodiments, a TRC-based approach is used to diagnose and monitormultiple diseases in a single reaction or test.

The methods described herein can be used, among other things, fordetermining the effect of a perturbation, including chemical compounds,mutations, temperature changes, growth hormones, growth factors,disease, or a change in culture conditions, on various target molecules,thereby identifying target molecules whose presence, absence or levelsare indicative of particular biological states. In one embodiment, someaspects described herein can be used to elucidate and discovercomponents and pathways of disease states. For example, the comparisonof quantities of target molecules present in a disease tissue with“normal” tissue allows the elucidation of important target moleculesinvolved in the disease, thereby identifying targets for thediscovery/screening of new drug candidates that can be used to treatdisease.

IV. KITS

The technology herein includes kits for evaluating the detection andquantitation of target nucleic acids in a sample. A “kit” refers to acombination of physical elements. For example, a kit may include, forexample, one or more components such as probes, including withoutlimitation specific primers, enzymes, reaction buffers, an instructionsheet, and other elements useful to practice the technology describedherein. The kits may include one or more TRCs of one or more of thetarget nucleic acids as described herein. These physical elements can bearranged in any way suitable for carrying out the present methods. Kitsmay include a preselected panel of TRCs or individually selected basedon application and preference.

Kits for detecting target nucleic acids may include, for example, a setof oligonucleotide probes. The probes can be provided on a solidsupport, as in an array (e.g., a microarray), or in separate containers.Kits can include further buffers, enzymes, labeling compounds, and thelike. Any of the compositions described herein may be comprised in akit. The kit may further include water and hybridization buffer tofacilitate hybridization of the two nucleic acid strands.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted (e.g., aliquoted into the wells of a microtiter plate). Wherethere is more than one component in the kit, the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a single vial. The kitsof the present disclosure also will typically include a means forcontaining the nucleic acids, and any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow molded plastic containers into which the desired vials areretained.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented. It is contemplated thatsuch reagents are embodiments of kits of the present disclosure. Suchkits, however, are not limited to the particular items identified aboveand may include any reagent used for the manipulation orcharacterization of the methylation of a gene.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, or other container means, into which acomponent may be placed, and preferably, suitably aliquoted. Where thereis more than one component in the kit, the kit also will generallycontain additional containers into which the additional components maybe separately placed. However, various combinations of components may becomprised in a container. The kits of the present disclosure also willtypically include a means for packaging the component containers inclose confinement for commercial sale. Such packaging may includeinjection or blow-molded plastic containers into which the desiredcomponent containers are retained.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the present disclosure, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the present disclosure.

Example 1—Generation of TRCs

TRCs were constructed from single-stranded DNA oligonucleotides(Integrated DNA Technologies). The 5′ and 3′ ends of theoligonucleotides were strategically placed within the toehold or bridgesequences and slow cooling under high-salt conditions forced the 5′ and3′ ends to sit adjacent and encouraged the correct intra-molecularligation by T4 DNA ligase (New England Biolabs). Treatment withexonucleases I and III eliminate unligated reactants. Gelelectrophoreses was used to confirm the correct size and purity of thefinal TRC population. Multiple TRCs were generated for each miRNA andthe final miRNA-specific TRC (i.e., one TRC per miRNA target) wasselected after empirical testing.

As a demonstration of feasibility, TRCs were designed and synthesizedtargeted toward miRNAs in the C. elegans let-7 family. let-7 familymembers are rigorously used to test miRNA platform specificity as theyfrequently differ by a single nucleotide. Here, two functional TRCs(FIG. 4) were used to distinguish and quantify miR-let-7a(UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 8) and let-7g(UGAGGUAGUAGUUUGUAUAGUU (SEQ ID NO: 9)); these miRNAs differ by a singlenucleotide at the 12th position (G>U, bolded and underlined). ThemiR-let-7a TRC had the sequence

(SEQ ID NO: 1) TAGTTCGCCCTACCTCCACATCCTCCACAAGCTATCCCCCGAACTATACAACCTACTACCTCACCGTTGTA,with CTACTACCT (SEQ ID NO: 2) as the toehold. The miR-let-7g TRC had thesequence

(SEQ ID NO: 5) TAGTTCGCCCTACCTCCACATCCTCCACAAGCTATCCCCCGAACTATACAAACTACTACCTCACCGT TGTA.

The in silico design process utilized various bioinformaticsapplications (IDT OligoAnalyzer, VIennaRNA, and NuPack) to model toeholddynamics of various TRC architectures. Achieving favorable parametersfor specific miRNA TMSD requires balancing thermodynamic properties(i.e., T_(m), ΔG, GC content of toehold sequence) with reactionconditions such as TRC and miRNA concentrations, salt concentration, andtemperature. Furthermore, bridge, probing, and backbone TRC sequenceswere stringently designed to have minimal hybridization capacity withnon-specific nucleic acids. Careful design of these sequences wasnecessary in order to eliminate false positive results brought on bypromiscuous binding of non-targeted nucleic acids. Similar unaligned,alien sequences are available through the NIST External RNA ControlsConsortium.

Promising candidate TRCs were synthesized and heuristically tested toidentify sensitive and specific constructs. Preliminary data wasobtained from 100 uL isothermal reactions (90 μL component mix plus 10μL input) started and maintained throughout at 37° C. φ29 polymerase wasselected for its high processivity in the elected temperature range,high fidelity, and robust strand-displacing properties. Significantalterations were made to the standard φ29 buffer conditions to achievehigh sensitivity and specificity. All reactions were performed for 60minutes in duration.

Molecular beacons, which are self-quenching hairpin probes containing afluorophore on the 5′ end, were utilized to quantify TRC RCR products.These beacons bind to the replicated products in a similar manner as theenvisioned fluorescent probes but permit rapid quantitation on amicroplate reader to investigate initial specificity and sensitivity. Asshown in

FIG. 2A, the incorporation of a toehold structure directed toward thetarget miRNA endows individual TRCs with exceptional specificity. Acircular, single-stranded miR-let-7a TRC-control (no toehold present)displays very little ability to discriminate between miR-let-7a andmiR-let-7g with only a 1.5 fold discrimination factor (middleconstruct). The toehold containing TRCs however, directed at eithermiR-let-7a or miR-let-7g, display discrimination factors (DF) of 71.2and 32.6 over their counterpart miRNA, respectively. This metricsurpasses the stated specificity of other commercially availablesingle-molecule platforms and suggests that with careful design, TRCscan be finely tuned to distinguish between highly similar miRNAmolecules. Furthermore, the sensitivity of the miR-let-7a TRC, as shownin FIG. 2B, indicates that this approach has the ability to quantitatemiRNAs at exceptionally low concentrations, even in a background ofuntargeted miRNAs. In this assay, miR-let-7a was present at 10 pM whilemiR-let-7g was present at 1 nM (100 fold excess). The miR-let-7a TRCstill achieves a robust β factor of 72.3; the β factor accounts foruntargeted background. As the microplate reader approach may beunderpowered; the platform take advantage of high-power fluorescentimaging to interrogate each individual replicated TRC on high-densityordered arrays, permitting far more sensitive quantitation at thesingle-molecule level.

Example 2—TRC Analysis of Circulating Pancreatic Cancer miRNA Signatures

Pancreatic cancer (PaCa) is a fundamentally challenging malignancy tocombat. Most cases are diagnosed in advanced stages with few effectivemedical options, and survival rates are abysmally low (5-20% over fiveyears). Many recent studies have outlined the utility of miRNAs, andspecifically cell-free miRNAs, in the course of PaCa for early detectionand disease surveillance.

TRC platform functionality is demonstrated by targeting nine miRNAspecies that have been detected at elevated levels in the plasma ofpancreatic cancer patients by currently available methods. These miRNAsinclude miR-10b, miR-21, miR-25, miR-106b, miR-155, miR-196a, miR-210,miR-212, and miR-221. The TRC platform is further validated by two setsof control TRCs to enable normalization across samples and confirm assayfunctionality including four miRNA targets present in the plasma ofhealthy humans miR-15b, miR-16, miR-24, miR-217 and three miRNA targetspresent only in C. eleganswith no sequence similarity to the humangenome cel-miR-39, -54, -238. Control TRCs are designed according to thesame process as target TRCs. The TRCs are constructed as described inExample 1.

In order to distinguish the products of 16 target miRNA TRCs inmultiplex, two additional independent, probing sequences areincorporated into the TRC, each with the potential to bind one of fourAlexaFluor labelled beacons. The two-color combination and order ofprobes binding to any particular nanosphere is unique and indicative ofa particular miRNA.

Following replicated TRC production by RCR and the formation ofreplicated nanospheres from the various multiplex miRNA testingpopulations, the products will be pipetted individually on to separateordered arrays. A brief incubation period permits replicated nanospheresto adequately bind to the functionalized surface. Arrays are thenprepared for probe hybridization by rinsing in a neutral pH buffer toremove contaminants from the initial reaction.

In order to approximate the significant background populationsencountered in actual plasma samples, size selected RNA populations areisolated from commercially available donor plasma samples usingcommercially available reagents and protocols (Qiagen). Prior to smallRNA isolation, plasma samples are spiked with mixed populations ofTRC-targeted miRNAs. Such populations include titrations (1 nM to 1 fM)of all target miRNAs, human control miRNAs and C. elegans controlmiRNAs.

Following targeted miRNA spike-in, the entire population of short RNApresent in the plasma (human and microbial RNA, ncRNAs, target miRNAs)is extracted, purified, and quantified using the optimized array-basedapproach.

Example 3—TRC Diagnostic Analysis of Oral Pathogenic Nucleic Acids

Multiplex pathogen detection of HIV, HSV-1, and EBV viral sequences isachieved by several TRC configurations (FIGS. 1A and 1E). TRCs areconstructed from several ligated oligonucleotides under diluteconditions and complimentary junction-spanning RNA splints and SplintRLigase, obtained from New England Biolabs, are added to encourageintra-molecular ligation. Multiple TRCs are generated specific for eachpathogen, and these may be used singly or in combination.

To transform these target sequences into functional primers that mayinitiate rolling circle replication (RCR), the overhanging 3′-endsequences were removed up to the double-stranded complimentary regionand present a 3′-OH group. This method, outlined in FIG. 1D, leveragesthe intrinsic 3′-exonuclease (3′-exo) activity exhibited by DNApolymerases or a separate standalone exonuclease. The inherent 3′-exoactivity of φ29 DNA polymerase was shown to remove the 3′-overhangingssDNA and ssRNA, albeit with different efficiencies from hybridizedtarget nucleic acids and initiate RCR. Similar to φ29 DNA polymerase,Exonuclease T displays different efficiencies in digesting ssDNA andssRNA 3′-overhanging sequences. Both DNA polymerases possess isothermal,high processivity, and strand-displacing qualities with Bst DNApolymerase having the added advantage of thermostability, and thuspotentially higher assay specificity. The intrinsic properties of theseDNA polymerase are important factors that contribute to the efficientand robust creation of replicated nanospheres.

Synthesized oligonucleotides (ssDNA, ssRNA, and dsDNA) with varyingdegrees of mismatching to the TRC pathogen sequence regions are used toconfirm hybridization, priming, exonuclease resection of the3′-overhanging sequences and the initiation of DNA synthesis throughRCR.

Successful reactions produce high-molecular weight (>70 kb) DNA productsthat are confirmed through gel electrophoresis. RCR reactions areevaluated using molecular beacons, which target accessory sequences onthe replicated nanospheres, using a fluorescent plate reader (FIG. 1B).Following replicated nanosphere creation by RCR from the variouspopulations, the products will be pipetted individually on to differentpatterned arrays. Arrays are then prepared for probe hybridization byrinsing in a neutral pH buffer to remove contaminants from the initialreaction.

Fluorescent probes are used to interrogate the bound replicatednanospheres. Four different and distinct fluorescent probes areutilized, one color for all replicated nanospheres produced from TRCstargeting a given specific pathogen (HIV, HSV-1, and EBV) or thepositive control TRC. Washed arrays containing fluorescentprobe-hybridized replicated nanospheres are imaged using anepi-fluorescence detection microscope. The number of pathogen-specificfluorescence signals is significantly correlated with targetoligonucleotide concentrations. Control and pathogen titration seriesand mixed oligonucleotide populations indicate any platform bias.

Pathogenic saliva samples are prepared (˜200-400 μL) from artificialand/or commercially obtained donor saliva spiked with variousconcentrations of commercially available viral mimics of HIV, HSV-1 andEBV (FIG. 1E). The viral mimics, available from AcroMetrix and used asdiagnostic controls, represent, intact, encapsidated viral particles.The artificial mimic pathogens are initially obtained as a stocksolution of viral particles in plasma. This is further diluted in thesaliva according to a titration series (10¹⁰ viral particles, 1.0 logsteps). All samples contain the control TRCs and target oligonucleotide.Bulk nucleic acids present in the saliva (human and microbial DNA andRNA, HIV RNA, HSV-1 and EBV DNA, control oligonucleotide) are extracted,purified, and quantified according to previously outlined methods andcommercially available reagents (e.g., MagJET Viral DNA and RNA kit,μDrop DNA quantification plate, Thermo Scientific).

Significant importance should be placed on obtaining a digital,quantified population assessment that reflects the expected viral load.System biases and inefficiencies may be apparent using the surrogateoral biospecimens and platform adjustments will be made accordingly.

The limits of detection based on previously collected data may placevalues at: HIV infected individuals may have as few as 100 copies/mL inthe saliva; HSV-1 in saliva oral swabs may be present at 1,000copies/mL, but can be detected at 10-fold lower concentrations; EBV hasbeen reported at considerably higher levels, approaching 10⁵ copies/mL.

Example 4—TRC Diagnostic Analysis of Oral Pathogenic Nucleic Acids

The diagnostic platform is used to interrogate microRNA populations withimmediate utility for T1D DR. 30 miRNAs were selected with significantassociation to DR initiation and progression in patient populations(FIG. 3). To normalize across samples and confirm assay functionality,two sets of controls are utilized: (i) two diabetic non-responsive miRNAtargets present in the biofluids of all humans (miR-15b and miR-16) and(ii) two miRNA targets present only in C. elegans with no sequencesimilarity to the human genome (cel-miR-39 and miR-54). The latter isspiked in to samples (both surrogate and clinical) to control forrun-specific variation and isolation efficiency.

Following TRC validation, replicated nanospheres will be randomly loadedonto functionalized, high-density ordered arrays. Fluorescent probes areused to interrogate replicated nanospheres at this stage.High-resolution imaging is performed using a Nikon Ti-E epifluorescencemicroscope. Stitching and analysis of the four-color array images iscarried out using Nikon Elements software and an in-house deconvolutionprogram based in ImageJ. Incorporation of software (e.g., BlobFinder)may aid in identifying and defining individual nanospheres. The probehybridization reactions and image collection are automated with standardmicrofluidic devices and modifications to the existing microscope.

Small RNA populations isolated from commercially available surrogateplasma, saliva, and urine specimens recapitulate the significantbackground nucleic acid populations encountered in patient samples.These biofluids are spiked with mixed populations of TRC-targetedmiRNAs. Such populations include titrations (1 fM to 1 nM) of all targetmiRNAs, human control miRNAs and C. elegans control miRNAs. Sizeselected RNA populations are isolated using commercially availablereagents and protocols (e.g., Qiagen miRNeasy). The entire population ofsmall RNAs present in the specimen (human and microbial RNA, ncRNAs,target miRNAs) is extracted, purified, and quantified using theoptimized and automated approach.

The platform is evaluated using a cohort of 30 to 50 clinically relevantDR patient samples. Leveraging the platforms high-throughput screeningcapabilities, the platform is used to characterize the miRNA populationsin patient samples. The data from 50 targets is used to yield a highlycorrelative miRNA signature for DR risk and severity as well as analysisof archived samples for patients that went on to develop DR and samplesshowing rapid progression to vision loss.

Example 5—TRC Technology Applied to Bead-Based Detection

Two sets of miRNA targets are used to assess the Luminex technologyusing surrogate plasma samples. First, a group of ten control C. elegansmiRNA targets with no sequence similarity to the human genome but withhigh sequence homology to each other is used to assess workflowefficiency, control for run-to-run variation, define the limits of TRCspecificity, sensitivity, and dynamic range. Furthermore, the C. eleganscontrols are used in constructing a standard curve for quantification. Asecond group of ninety miRNA targets are used to prove the robust natureof the design application and the platform's ability to quantitatepotential real-world miRNA biomarkers. This second set of targets isderived from the NanoString nCounter miRNA Discovery panel to facilitatecomparison.

Commercially available, healthy human plasma are used as a surrogatestarting material. Fifty surrogate samples are spiked with variouscombinations of the ten C. elegans control miRNAs representingtitrations ranging from 1 fM to 1 nM.

Two input methods are analyzed: crude plasma samples and isolated,size-specific small RNA populations. The entire population of small RNAs(<200 bp) present in the specimen (human and microbial RNA, ncRNAs,target miRNAs, etc.) is extracted, purified, and quantified using thestandard reagents and protocols (i.e., Qiagen miRNeasy). The nucleicacid populations in plasma recapitulates the significant backgroundencountered in patient samples.

The bioinformatics pipeline is employed to design and construct a uniqueTRC for each of the ten C. elegans and 90 native target miRNAs. LuminexMagPlex beads contain carboxyl groups to facilitate the attachment ofnumerous macromolecules, including nucleic acids. As TRCs are circularin nature, amino-modifier C6 dT phosphoramidates are used duringsynthesis of TRC oligonucleotides for coupling to Luminex beads.TRC-specific, complementary, secondary primers are also attached to theLuminex beads to further extend the unique replicated TRC products,covalently attaching such products to the beads. This process results ina highly-intertwined but discrete, bead-product detectable on theLuminex 200 platform (FIG. 6).

Functionalized microspheres are purified by magnetic isolation andcombined into a single reaction tube. RCR reaction components (buffer,nucleotides, polymerase, microspheres) are combined with a crude orpurified surrogate sample and incubated under isothermal conditions. Thereplicated product of an individual TRC RCR reaction is a single, longnucleic acid composed of hundreds of concatenated and complimentary TRCsequences. To covalently attach the RCR product, a secondary primer isemployed in the strand-displacing reaction; this effectively inducesrolling circle amplification (RCA) (FIG. 6). Microspheres and the boundRCR/RCA product are isolated using magnetic separation.

To label replicated TRC products for detection on Luminex platforms,fluorescently-labelled nucleotides (i.e., Fluorescein-12-dCTP;PerkinElmer) are utilized. Previous experience with Bst and Bsupolymerases have demonstrated efficient incorporation of such modifiednucleotides in RCR. Alternatively, an intercalating dye such as Sybrcould be used to detect replicated TRCs bound to beads.

Microsphere analysis is carried out according to the standard operatingprotocol provided by Luminex. The Luminex 200 instrument is capable ofsimultaneously analyzing up to 100 unique reactions (i.e., 100 miRNAtargets) with more advanced versions of the technology capable ofhandling up to 500 unique reactions. Luminex's xPONENT software is usedfor continuous monitoring and quantification of all target miRNAs.

Example 6—TRC Technology for Research and Diagnosis of Endometriosis

An endometriosis mouse model is used to assess TRCs as a viable researchtool and clinical diagnostic test. Endometriosis is induced bytransplanting endometrial fragments from Luciferase/GFP donor mice,allowing for live monitoring and imaging of disease progression. A listof 25 miRNAs with significant association to the presence ofendometriosis in patient populations was developed (FIG. 7).

To normalize across samples and confirm assay functionality, each TRC isassayed against the target miRNA from the library and biologicallysimilar or synthetically similar miRNA target. These biologicallysimilar targets may differ by 1 or 2 base-pairs. If no biologicallysimilar miRNA exists, then a synthetic target is used.

Interrogation of specificity and sensitivity is performed in three ways.First, similar to Example 1, specificity is verified with thefluorescent microplate reader, which provides simple output data todetermine if the TRC needs to be redesigned for its miRNA target. Once atarget is verified on the microplate reader, the reaction conditions forqRT-RCR are determined (FIG. 7). Lastly, the TRC qRT-RCR is compared tothe sensitivity and specificity of current miRNA qRT-PCR detectionprotocols (FIG. 7), providing a direct comparison of the presenttechnology to the current gold standard for clinical diagnostic labs.

The endometriosis miRNA signature detection capabilities of the TRClibrary is determined in 5 endometriosis-induced mice and 5 controlmice. To confirm noninvasive growth of ectopic lesions in mice withendometriosis, Luciferase (luc)/GFP mice [FVB-Tg (CAG-luc,-GFP)L2G85Chco/FathJ mice, Jackson Laboratory] are employed. In brief,endometrial fragments isolated from Luc/GFP donor mice are implantedinto wild type recipient mice (non-transgenic littermates) (FIG. 7).

Endometriosis is induced in 6-week-old female mice and allowed toprogress for two weeks. After two weeks, vaginal cells are isolated fromendometriosis-induced mice and histologically examined every day for asubsequent two weeks. These cells are stained with Crystal Violet todetermine the estrus cycle of each mouse. Endometrial tissue and bloodis harvested at the proestrus stage of each mouse. Bioluminescenceimaging of the ectopic lesions in each mouse is captured once a week totrack lesion growth and development.

All mice are euthanized in compliance to American Veterinary MedicalAssociation protocols. From these harvested samples, miRNA is isolatedand interrogated for the presence of the endometriosis TRC library.

The sensitivity and specificity of a subset of control TRCs is evaluatedin a small set (five [5] healthy, endometriosis-free patients) ofclinical samples from women (FIG. 6). These clinical samples are bothuterine tissue and plasma taken from the same patient to correlate theamount miRNA detected in each biospecimens.

miRNA will be isolated from both sets of biospecimens and evaluatedusing TRCs that target miRNAs that are present in healthy individualsand have no disease indication (FIG. 7).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of the present disclosurehave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe methods and in the steps or in the sequence of steps of the methodsdescribed herein without departing from the concept, spirit and scope ofthe present disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the present disclosure asdefined by the appended claims.

1-78. (canceled)
 79. A method for determining the presence or theabsence of a pathogenic nucleic acid in a sample comprising: (a)obtaining a target reporter construct (TRC); (b) contacting the targetreporter construct (TRC) with a sample to form a hybridized TRC if apathogenic nucleic acid is present in the sample; (c) performing rollingcircle replication (RCR) on the hybridized TRC, thereby obtaining areplicated TRC if a pathogenic nucleic acid is present in the sample;(d) contacting the replicated TRC with a detectable moiety, wherein thedetectable moiety binds a probe sequence of the replicated TRC to form abound detectable moiety if a pathogenic nucleic acid is present in thesample; and (e) detecting for the bound detectable moiety, wherebydetection of the bound detectable moiety corresponds to the presence ofthe pathogenic nucleic acid in the sample and the failure to detect thebound detectable moiety corresponds to the absence of the pathogenicnucleic acid in the sample.
 80. The method of claim 79, wherein thesample is further defined as a biological sample.
 81. The method ofclaim 80, wherein the biological sample is blood, plasma, saliva, orurine.
 82. The method of claim 80 or 81, wherein the biological samplecomprises a pathogenic species.
 83. The method of claim 82, wherein thepathogenic species is a virus, bacteria, or fungus.
 84. The method ofclaim 83, wherein the bacteria is Chlamydia trachomatis, gonorrhoea, orTreponema.
 85. The method of claim 79, wherein the detectable moiety hasbeen conjugated to a colloidal particle.
 86. The method of claim 79,wherein the sample comprises a population of pathogenic nucleic acids.87. The method of claim 86, wherein the pathogenic nucleic acids withinthe population hybridize to a complementary target sequence of the TRC.88. The method of claim 87, wherein the pathogenic nucleic acids is asmall RNA.
 89. The method of claim 88, wherein the small RNA is lessthan 200 nucleotides in length.
 90. The method of claim 88, wherein thesmall RNA is a microbial RNA.
 91. The method of claim 79, wherein theTRC is attached to a solid substrate.
 92. The method of claim 91,wherein the solid substrate is a test strip.
 93. The method of claim 79,wherein the TRC is a closed, partially single-stranded nucleic acidcomprising: (a) one or more target sequences complementary to pathogenicnucleic acids, wherein the target sequences are toehold switchescomprising a short duplex structure with an overhang; (b) a bridgesequence forming a double-stranded portion of the TRC, wherein thebridge sequence comprises a stabilizing sequence for the formation ofnanospheres during replication; and (c) an accessory sequence comprisinga multifunctional probe sequence.
 94. The method of claim 79, whereinthe TRC is between 50 and 100 nucleotides in length.
 95. The method ofclaim 79, wherein performing RCR comprises introducing astrand-displacing polymerase.
 96. The method of claim 95, wherein thestrand-displacing polymerase is φ29, Bst, or Bsu.
 97. The method ofclaim 79, wherein step (b) and step (c) are performed on a singlesubstrate.
 98. The method of claim 79, wherein RCR is performed for 45minutes to 90 minutes.
 99. The method of claim 79, wherein detecting thedetectable moiety comprises normalizing to a control replicated TRC.100. The method of claim 79, wherein the method is further defined as apoint of care assay.
 101. A method for detecting a pathogenic nucleicacid in a sample comprising: (a) obtaining a target reporter construct(TRC); (b) contacting the target reporter construct (TRC) with a sampleto form a hybridized TRC; (c) performing rolling circle replication(RCR) on the hybridized TRC, thereby obtaining a replicated TRC; (d)contacting the replicated TRC with a detectable moiety, wherein thedetectable moiety binds a probe sequence of the replicated TRC to form abound detectable moiety; and (e) detecting the bound detectable moiety,thereby detecting the pathogenic nucleic acid in the sample.